Methods of generating glial and neuronal cells and use of same for the treatment of medical conditions of the cns

ABSTRACT

A method of generating neural and glial cells is provided. The method comprising growing human stem cells under conditions which induce differentiation of said human stem cells into the neural and glial cells, said conditions comprising the presence of retinoic acid and an agent capable of down-regulating Bone Morphogenic Protein activity.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of generating glia cells suchas oligodendrocyte like cells and the use of same for the treatment ofmedical conditions of the CNS.

Myelin, the fatty substance which encloses certain axons and nervefibers, provides essential insulation, and enables the conductivity ofnerve cells which transmit electrical messages to and from the brain.

Aberration in myelination can lead to several pathologies in the centralnervous system. These include for example, autoimmune diseases (e.gMultiple Sclerosis), congenital leukodystrophies (e.gPelizaeus-Merzbacher, vanishing white matter, adrenoleukodystrophy),infectious diseases (e.g. progressive multifocal leukoencephalopathy,postinflammatory demyelinated lesions), neurodegenerative diseases (e.g.multisystem degeneration), vascular diseases (vascularleukoencephalopathies, subcortical infarcts), congenital genetic defects(e.g. amyotrophic lateral sclerosis [ALS], Alzheimer disease, Parkinsondisease) and brain and spinal cord trauma or injuries which aredemyelinative and possibly neoplasms (e.g. oligodendrio-glioma).

Treatment of these pathologies may be effected by cell replacementtherapies which eventually may allow achieving regional or even globalrepair of myelin as indicated by experiments in MBP-deficient shiverermice (e.g., Yandava et al., 1999).

Oligodendrocytes extend as many as 50 processes which wrap around axonsto form myelin sheaths. In vitro and in vivo, the development ofoligodendrocytes proceeds in steps from neural stem cells to bipolarprogenitors, then to multipolar precursor cells having several mainprocesses which subsequently arborize and form the multiple branches ofthe mature cells (Rogister et al., 1999; Grinspan, 2002). Earlyoligodendrocytes precursors (OP) express the PDGF-receptor and later thespecific O4 sulfatide marker of pre-oligodendrocytes which persists inramified immature oligodendrocytes, then the maturating cells expressgalactocerebrosides (O1, GalC) and 2′,3′ cyclic nucleotide 3′phosphodiesterase (CNP), and at the final stage become postmitoticmature oligodendrocytes that synthesize the myelin membrane componentssuch as proteolipid protein (PLP) and myelin basic protein (MBP).

Embryonic stem (ES) cell lines being amenable to mass culture anddifferentiation into specific cell lineages in vitro are a potentiallarge scale source of oligodendrocytes for brain and spinal cordtransplantation, which has been studied in experimental models usingmouse ES cells (Liu et al., 2000; Zhang et al., 2004; Glaser et al.,2005; Zhang et al., 2006). For successful transplantation, the cellsneed the capacity to migrate into the CNS and, therefore, theappropriate stage of cell differentiation must be defined, since moredifferentiated oligodendrocytes migrate minimally but conversely thecells that migrate most are less likely to differentiate into maturemyelinating cells (Warrington et al., 1993; Foster et al., 1995; Yandavaet al., 1999).

Zhang et al (2004; 2006) describe a procedure which converts murine EScells into highly branched and ramified oligodendrocytes and producesprogenitor which upon implantation in shiverer mouse brain tissue,migrate and form dense arrays of myelinated nerve fibers. However, theneed for human ES cells which can be converted and expanded intofunctional oligodendrocytes is yet far from being fulfilled.

The availability of human ES cell (huESC) lines, derived fromsupernumerary human IVF blastocysts, provides a potential large scalesource of human oligodendroglial and neural precursor cells that couldbe used in clinical settings to treat a variety of severe humanneurological diseases, congenital or acquired. Neural stem cells frombrain or spine of aborted human fetuses may be another source of neuralstem cells, but preparing engraftable quantities of cells from fetal oradult human brain presents many problems (Goldman, 2005). While humanfetal brain cells can differentiate into ramified matureoligodendrocytes (Zhang et al., 2000), most studies show that fetalbrain precursor cells lose their ability to produce oligodendrocytesupon expansion (Chandran and Compston, 2005). Therefore, the use oflaboratory-established huESC lines that can be expanded in relativelylarge scale cultures, would be highly advantageous to prepare thegrafts.

Previous attempts to use human ES cells lines for derivingoligodendrocytes have yielded only partial success. A first studydescribed the preparation of neural tube-like rosettes which wereexpanded by bFGF into neurospheres and eventually transplanted into thethird ventricule of newborn mice brain (Zhang et al., 2001). In thiswork only elongated bipolar O4⁺ OPC were obtained and no myelination wasshown following transplantation. Another study (Reubinoff et al., 2001)produced neurospheres from huESC with EGF and bFGF, and showedmultipolar O4⁺ cells with a few processes, as well as CNP-positive cellsafter transplantation but without evidence for myelination. A furtherstudy, with huESC-derived neurospheres that were produced with bFGF andnoggin yielded O4⁺ precursors but no ramified or mature cells, and notransplantation was done (Itsykson et al., 2005).

The inability of neurons in the mammalian central nervous system (CNS)to regenerate axons is due to inhibitory influences by glial cells ofthe CNS, which prevent the re-activation of growth promoting genes.However, axonal regeneration occurs in the peripheral nervous system(PNS). Retinoic acid-mediated signal transduction is known to inducethese regenerative processes in the PNS. Retinoic acid (RA) is one ofthe active forms of vitamin A and is involved in life maintainingprocesses such as reproduction, embryonic development, vision, growth,cellular differentiation and proliferation, tissue maintenance and lipidmetabolism. By activating a number of regulatory genes and signalingmolecules, retinoic acid plays a crucial role during the development ofthe vertebrate nervous system. RA also initiates cell development ofimmature blood cells. One of the many medical uses of RA is for thetreatment of Acute Myeloid Leukemia. Administration of RA causes thehighly proliferative immature blood cells typical of this disease, todifferentiate and develop into functional cells.

Because of its known functions, retinoic acid was used to induce neuraldifferentiation and neurospheres in murine ES cells (Bain et al., 1995;Liu et al., 2000). Following this approach (Nistor et al., 2005),retinoic acid was used for the induction of neural-lineage cells fromHuESC, in concurrence with preferential selection ofoligodendrocyte-lineage cells by media components and matrigeladherence. Although “high purity functional oligodendrocytes from huESC”were claimed, no well differentiated, highly branched and ramifiedoligodendrocytes were produced, and after transplantation to spinal cordof shiverer mouse, there were only small patches of MBP-positive cellswith no evidence for extended areas of remyelination and no myelinatednerve fibers of at least 100 μm.

All the described attempts to produce fully differentiated and matureoligodendrocytes from human ES cells convey that procedures which havebeen successful with murine ES cells cannot be extrapolated to human EScells. Thus, the need for production of fully differentiated and maturehuman oligodendrocytes still exists.

For this purpose, key genes, encoding transcription factors which areobligatory for the development of the oligodendrocyte lineage need to bestudied, in order to define the agents that are needed to convert huESCinto oligodendrocytes, and the agents which delay this process, and needto be inhibited. For example, Bone Morphogenetic Proteins (BMPs) aregroup of growth factors known for their ability to induce the formationof bone and cartilage. Signal transduction through BMPs, is importantfor the development of the heart, central nervous system, and cartilage,as well as post-natal bone development. Noggin, which binds to membersof the TGF-β superfamily, plays a crucial role in bone development andneurulation, by regulating the functions of BMP's. For example, it wasfound that noggin counteracts the effect of BMPs, which in turn werefound to inhibit oligodendrocyte development from rat fetal brain(Mehler et al., 1997; Mehler et al., 2000).

There is thus a widely recognized need for producing fullydifferentiated, and mature oligodendrocytes propagated and expanded fromhuman ES cells, that addresses these deficiencies.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of generating neural and glial cells, the method comprisinggrowing human stem cells under conditions which induce differentiationof the human stem cells into the neural and glial cells, the conditionscomprising the presence of retinoic acid and an agent capable ofdown-regulating Bone Morphogenic Protein activity.

According to another aspect of the present invention there is provided amethod of generating glial cells, the method comprising: (a) growinghuman stem cells in the presence of retinoic acid, under conditionswhich allow formation of neurospheres; and (b) contacting theneurospheres with an agent capable of down-regulating Bone MorphogenicProtein activity thereby generating the glial cells.

According to yet another aspect of the present invention there isprovided a method of generating oligodendrocytes, the method comprising:(a) growing human stem cells in the presence of retinoic acid underconditions which allow the formation of neurospheres; and (b) contactingthe neurospheres with an agent capable of down-regulating BoneMorphogenic Protein activity thereby generating the oligodendrocytes.

According to still another aspect of the present invention there isprovided a method of generating neurons, the method comprising: (a)growing human stem cells in the presence of retinoic acid underconditions which allow the formation of neurospheres; and (b) contactingthe neurospheres with an agent capable of down-regulating BoneMorphogenic Protein activity under conditions which allow neuronal cellsgeneration, the conditions comprising Sonic Hedgehog (Shh), therebygenerating the neurons.

According to an additional aspect of the present invention there isprovided an isolated oligodendrocyte cell generated according to any ofthe above methods.

According to yet an additional aspect of the present invention there isprovided an isolated astrocyte cell generated according to any of theabove methods.

According to still an additional aspect of the present invention thereis provided an isolated neuronal cell generated according to any of theabove methods.

According to a further aspect of the present invention there is providedan isolated population of cells comprising human cells wherein,

(i) at least N % of the human cells comprise at least one matureoligodendrocyte phenotype; and

(ii) at least M % of the human cells comprise at least one stem cellphenotype.

According to yet a further aspect of the present invention there isprovided a method of treating a medical condition of the CNS in asubject-in-need thereof, the method comprising administering to thesubject a therapeutically effective amount of the cells of the presentinvention, thereby treating the medical condition of the CNS.

According to still a further aspect of the present invention there isprovided use of the cells of the present invention, for the manufactureof a medicament identified for treating a medical condition of the CNS.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe cells of the present invention and a pharmaceutically acceptablecarrier or diluent.

According to further features in preferred embodiments of the inventiondescribed below, the human stem cell is a human embryonic stem cell.

According to still further features in the described preferredembodiments the oligodendrocyte comprises a precursor oligodendrocytephenotype.

According to still further features in the described preferredembodiments the precursor oligodendrocyte phenotype comprises cellmigration.

According to still further features in the described preferredembodiments the precursor oligodendrocyte phenotype comprises cellexpansion.

According to still further features in the described preferredembodiments the cell expansion comprises in vitro cell expansion.

According to still further features in the described preferredembodiments the cell expansion comprises in vivo cell expansion.

According to still further features in the described preferredembodiments the precursor oligodendrocyte phenotype comprises a markerexpression selected from the group consisting of PDGF-receptor, O4sulfatide marker, galactocerebrosides (O1, GalC), Nkx2.2, Sox10,oligodendrocyte specific protein (OSP), myelin-associated glycoprotein(MAG), 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNP),glutathione-S-transferase (GST), adenomatous polyposis coli (APC);myelin oligodendrocyte glycoprotein (MOG), CNPase, MOSP andOligodendrocyte NS-1.

According to still further features in the described preferredembodiments the precursor oligodendrocyte phenotype comprises a cellmorphology selected from the group consisting of round or elongated,bipolar or multipolar shape.

According to still further features in the described preferredembodiments the oligodendrocyte comprises a mature oligodendrocytephenotype.

According to still further features in the described preferredembodiments the mature oligodendrocyte phenotype comprises in vivo andin vitro myelin production.

According to still further features in the described preferredembodiments the glial cells comprise astrocytes.

According to still further features in the described preferredembodiments the glial cells comprise mature oligodendrocytes whichcomprise a mature oligodendrocyte phenotype.

According to still further features in the described preferredembodiments the mature oligodendrocyte phenotype comprises a matureoligodendrocyte marker expression.

According to still further features in the described preferredembodiments the mature oligodendrocyte marker is selected from a groupconsisting of PLP, MBP, MAG and MOG.

According to still further features in the described preferredembodiments the mature oligodendrocyte phenotype comprises a matureoligodendrocyte structural phenotype.

According to still further features in the described preferredembodiments the mature oligodendrocyte structural phenotype is branchedand ramified.

According to still further features in the described preferredembodiments the method further comprising forming stem cell aggregatesprior to step (a).

According to still further features in the described preferredembodiments the conditions further comprise culturing the stem cells inthe presence of a growth factor.

According to still further features in the described preferredembodiments the growth factor is selected from a group consisting ofbFGF and EGF.

According to still further features in the described preferredembodiments a concentration of the EGF comprises a range of 10-40 ng/ml.

According to still further features in the described preferredembodiments the conditions further comprise culturing the neurosphereson an adherent substrate following step (a).

According to still further features in the described preferredembodiments the conditions further comprise culturing the neurosphereson a cationic substrate following step (a).

According to still further features in the described preferredembodiments the adherent substrate comprises a substrate selected fromthe group consisting of a matrigel and an extracellular matrixcomponent.

According to still further features in the described preferredembodiments the extracellular matrix component is selected from thegroup consisting of collagen, laminin and fibronectin.

According to still further features in the described preferredembodiments the cationic substrate is selected from a group consistingof poly D/L lysine and Polyornithine FN.

According to still further features in the described preferredembodiments a concentration of the retinoic acid comprises a range of1-50 μM.

According to still further features in the described preferredembodiments the retinoic acid is selected from the group consisting ofretinoic acid, retinol, retinal, 11-cis-retinal, all-trans retinoicacid, 13-cis retinoic acid and 9-cis-retinoic acid.

According to still further features in the described preferredembodiments the step (a) is effected for 20-30 days.

According to still further features in the described preferredembodiments the step (b) is effected for 6-10 days.

According to still further features in the described preferredembodiments the agent is selected from the group consisting of noggin,chordin, chordin like BMP inhibitor (CHL2), Neuralin, follistatin, GDF3,Crossveinless-2 (hCV-2), Ectodin, Sclerostin, connective tissue growthfactor (CTGF), BMP-3, Inhibin, Cerberus, Coco, PRDC, DAN, USAG1, Twistedgastrulation (TSG), gp130 signaling cytokines and gremlin.

According to still further features in the described preferredembodiments the agent is noggin.

According to still further features in the described preferredembodiments a concentration of the noggin comprises a range of 10-100ng/ml.

According to still further features in the described preferredembodiments step (b) is effected at least in part in the absence ofgrowth factors.

According to still further features in the described preferredembodiments step (b) is effected in the presence of laminin and vitaminC.

According to still further features in the described preferredembodiments the method further comprising:

dissociating cells of the neurospheres; and

passaging the dissociated cells; following step (a) and/or concomitantlywith step (b).

According to still further features in the described preferredembodiments the passaging is effected every 8-10 days.

According to still further features in the described preferredembodiments the method further comprising isolating a glial cellsubpopulation of interest following step (b).

According to still further features in the described preferredembodiments the method further comprising isolating the oligodendrocytesfollowing step (b).

According to still further features in the described preferredembodiments the method further comprising isolating the neuronsfollowing step (b).

According to still further features in the described preferredembodiments the medical condition is selected from the group consistingof autoimmune diseases, Guillan-Barre syndrome or congenitalleukodystrophies, adrenoleukodystrophies, Pelizaeus-Merzbacher,Charcot-Marie-Tooth, Krabbe or Alexander disease, vanishing white mattersyndrome, progressive multifocal leukoencephalopathy, infectiousdemyelinating diseases, postinflammatory demyelinated lesions,neurodegenerative diseases, multisystem degeneration, vascular diseases,ischemic white matter damage, vascular leukoencephalopathies,subcortical infarcts, brain trauma, spinal cord trauma, demyelinativeinjury, neoplasms and oligodendrio-glioma.

According to still further features in the described preferredembodiments the cells comprise oligodendrocytes and the medicalcondition is associated with insufficient myelination According to stillfurther features in the described preferred embodiments the cellscomprise neuronal cells and the medical condition is selected from thegroup consisting of motor neuron diseases, progressive muscular atrophy(PMA), spinal muscular atrophy (SMA), progressive bulbar palsy,pseudobulbar palsy, primary lateral sclerosis, neurological consequencesof AIDS, amyotrophic lateral sclerosis (ALS), Alzheimer's disease,developmental disorders, epilepsy, multiple sclerosis, neurogeneticdisorders, Parkinson's disease, neurodegenerative disorders, stroke,spinal cord injury and traumatic brain injury.

According to still further features in the described preferredembodiments the cells comprise astrocytes and the medical condition isselected from the group consisting of Alexander disease, epilepsy,Alzheimer's disease, spinal cord injury, traumatic brain injury andneurogenesis deficiencies.

According to still a further aspect of the present invention there isprovided a method of determining an effect of treatment on neural cellfunctionality, the method comprising: (a) subjecting cell generatedaccording to any of the above methods to the treatment; and (b)determining at least one of a structural or functional phenotype of thetreated cell as compared to an untreated cell, thereby determining aneffect of the treatment on neural cell functionality.

According to still further features in the described preferredembodiments the treatment comprises a treatment with a drug.

According to still further features in the described preferredembodiments the treatment comprises a treatment with a condition.

According to still further features in the described preferredembodiments the condition is selected from the group consisting of anelectrical treatment and an irradiation treatment.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel methods of generatingglia cells and in particular oligodendrocytes and methods of using suchcells for the treatment of medical conditions of the CNS.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-h depict the early steps (as described in Example 1 in theExamples section, and summarized in Table 1, condition 1) in obtainingneuroglial sphere cells (NSc) from human ES cells, and theirdifferentiation into O4⁺ oligodendrocytes in response to noggin; FIG. 1a shows RT-PCR images depicting the gene expression profile of retinoicacid treated neurospheres at the end of step R of the treatment (10 μMRetinoic acid treatment; lane 1) and at the end of step S (Suspensionculture, where neurospheres are allowed to ripen), without noggin (lane2) or with noggin (50 ng/ml Noggin-Fc chimera; lane 3). Note thatretinoic acid and noggin are both needed in order to induce theexpression of genes characterizing oligodendrocyte development: Nkx2.2,Sox10, Olig2, Olig1. FIG. 1 b shows RT-PCR images depicting the geneexpression profile of FGF treated neurospheres (left lane), andneurospheres additionally treated with thyroid hormone (triiodothyronin,T3; right lane). Note that this procedure, which was found to besuccessful for murine ES cell differentiation into oligodendrocytes,does not appear to induce Nkx2.2 in human ES cells. FIG. 1 c showsRT-PCR images depicting the expression profile of Bone MorphogeneticProtein (BMP) genes by human ES cells, before (lane 1) and after (lane2) step R (10 μM Retinoic acid treatment), after step S (Suspensionculture, where neurospheres are allowed to ripen), with noggin (50 ng/mlNoggin-Fc chimera; lane 3) or without noggin (lane 4), after step M(adherence to matrigel-coated plates of neurospheres from cultureswithout noggin at step S; lane 5), same after step F1 (5 ng/ml EGF andbFGF, 50 ng/ml Noggin-Fc chimera, 1 μg/ml mouse laminin, and 50 μg/mlVit C) and step F2 (same but without EGF, bFGF; lane 6). Note expressionpanel shows several BMP genes (inhibitors of oligodendrocytedevelopment) are induced by retinoic acid treatment and some of themincrease further during the differentiation procedure. Noggin (a BMPfamily antagonist), is required to counteract BMPs induction by RA. FIG.1 d is a fluorescent light micrograph depicting immunostainedneurospheres during step M (adherence to matrigel). Immunostaining showsthat after only 4 days in step M the outgrowth from the neurospherescontain neurons (immunostained for neuronal tubulin-βIII, red) and smallcells stained for the oligodendrocyte-specific marker O4 (green). Sizebar, 20 μm. FIG. 1 e is a fluorescent light micrograph depicting the O4positive cells (indicated by arrows) at the end of step M (after 9 days;panel 1). Cells show positive immunostaining for the astrocytes lineagemarker GFAP (panel 2) and are small unipolar cells (panel 3), whichidentifies them as glial progenitor cells. Size bar, 20 μm. FIG. 1 f isa fluorescent light micrograph depicting neurospheres after step F2(done on matrigel, without GF but with noggin, added only at this step,for 6 days). Immunostaining for PDGF Receptor-α (red) shows the presenceof elongated bipolar oligodendrocyte progenitors (OP). Immunostainingfor the oligodendrocyte-specific marker O4 (green) shows differentiatedhighly branched O4⁺ oligodendrocytes. Size bar, 20 μm. FIGS. 1 g-h arefluorescent light micrographs depicting the same culture described inFIG. 1 f, following 10 days without GF, without (FIG. 1 g) and with(FIG. 1 h) noggin. Immunostaining shows that without noggin there aremainly O4⁺ bipolar precursors, whereas with noggin there are numeroushighly branched well-differentiated oligodendrocytes. Size bar, 100 μm.

FIGS. 2 a-g depict mature myelin basic protein (MBP)-positiveoligodendrocytes differentiated from the human ES cells followingculture conditions as described in Table 1, condition 1, in Example 1 inthe Examples section. FIGS. 2 a-b are fluorescent light micrographsshowing the same immunostained cells after step F2 (10 days without GF)with noggin added only at steps F1,F2, and stained for MBP (red; FIG. 2a), or double-stained for O4 (green) and MBP (red; FIG. 2 b). FIGS. 2c-f are fluorescent light micrographs of different MBP-positive cellsimmunostained for MBP and O4. FIG. 2 c shows cells which are MBP and O4positive. FIGS. 2 d-e show that while some cells are still only O4positive (FIG. 2 d) some have matured further to the MBP⁺, O4⁻ phenotype(FIG. 2 e; Size bar for. FIGS. 2 c-e, 20 μm). FIG. 2 g is a bar graphdepicting quantitation assessment of the number of MBP⁺ cells in fields(such as in FIG. 2 f, Size bar, 100 μm). Note the strong stimulationcaused by noggin as compared to cultures without noggin (NT).

FIGS. 3 a-d are fluorescent light micrographs showing that human EScell-derived dissociated neuroglial sphere cells (huEs-NSc) cells can bedissociated by trypsin, expanded with EGF and bFGF on poly-D-lysinecoated plates and then differentiated into large matureoligodendrocytes. FIG. 3 a depicts cells grown in culture conditions asdescribed in condition 3 in Table 1, Example 1 in the Examples sectionafter step D3 (Expansion of huEs-NSc) with noggin. The cells werestarved of GF for 1 day, trypsinized and plated for 18 hours beforefixation and immunostaining for O4 (red) and dapi (blue). Comparisonwith dapi-stained nuclei shows a homogenous population of bipolarprecursors. Size bar 100 μm. FIGS. 3 b-c depict the accumulation ofmultibranched ramified oligodendrocytes in the same culture described inFIG. 3 a (Passage 3) after differentiation step F2, 10 days without GFbut with noggin. Higher magnification (FIG. 3 c) shows the branchingnetwork and the development of flat and broad membranes (arrows). Sizebar 100 μm. FIG. 3 d depicts the same culture described in FIGS. 3 a-c,immunostained for the O1 marker denoting mature oligodendrocytes (green)and for GFAP to visualize astrocytes. Size bar 100 μm.

FIGS. 4 a-f depict the in vivo myelinating capacity of human ES cellderived neuroglial sphere cells (HuES-NSc) injected into brain ofshiverer mice, which lack a functional gene for MBP. Any MBP stainingseen is, therefore, the result of myelin synthesis by the exogenouslytransplanted cells. One month following HuES-NSc transplantation, brainsections were immunostained for MBP (red) and nuclei was visualized bydapi (blue). FIG. 4 a-d,g are fluorescent light micrographs depictingsections of brain implanted with HuES-NScs, treated (FIGS. 4 b-d,g), ornot treated (FIG. 4 a) with noggin for 9 days at step D3 (passage step,as described in Table 3). FIG. 4 a-b are sections of the cortex. Notemore MBP stained fibers were observed with HuES-NSc treated with noggin(FIG. 4 b), and long fibers were seen (indicated by arrows). FIG. 4 e isa bar graph showing number of MBP fibers in cells cultured with (red) orwithout (blue) noggin. Note increased number of MBP fibers/field withnoggin. FIG. 4 c depicts MBP stain of a section field that includes partof the ventricule (v), visualizing the synthesis of MBP along theventricule walls (as indicated by an arrow). Arrowhead demonstrates howthe cells migrated into the brain parenchyma where MBP⁺ fibers ofvarious lengths are observed over a large area. FIG. 4 g shows the samefield stained for Human Nuclear Antigen (HNA, green) confirming thehuman origin of the myelinating cells. Size bar 100 μm. FIG. 4 d depictsan adjacent area in the striatum, stained for MBP (red) and nuclear dapi(blue). Note a dense cluster of MBP stained fibers in the center, witharrays of parallel fibers (as indicated by an arrowhead) and long fibers(as depicted by an arrow). Size bar 100 μm. FIG. 4 f shows nuclear dapistain of the areas seen in FIG. 4 c and 4 d, with ventricule (v), arrowand arrowhead depicting ventricle walls and migration into parenchyma,as in 4 c. Star indicates the center of field in FIG. 4 d. Size bar 100μm.

FIGS. 5 a-c,h are fluorescent light micrographs depicting themyelinating capacity of HuES-NSc in organotypic cultures of shiverermouse (lacking a functional gene for MBP) brain slices. FIGS. 5 a-cdepict a brain section following implantation of HuES-NSc, and stainedfor MBP (FIG. 5 a, c) and dapi (FIG. 5 b), as described in FIG. 4 a.FIG. 5 a depicts MBP stained fibers (as indicated by arrows), in anextended area of the entorhinal cortex, two weeks after implantation inthe hippocampal region. FIGS. 5 b depicts cell nuclei stained with dapi.Size bar 100 μm. FIG. 5 c shows, in higher magnification, dense arraysof MBP-stained fibers, some of extended length (indicated by arrows).FIG. 5 h is the same section of FIG. 5 c, stained with nuclear dapi.Size bar 100 μm. FIGS. 5 d-f are electron micrographs of sections in thecorpus callosum region, depicting nerves. FIG. 5 d depicts dysmyelinatednerves in the Shiverer brain, whereas in brain slices transplanted byhuES-NS cells treated with noggin (as above), compact myelin with majordense lines is clearly observed (as indicated by arrows in FIGS. 5 e and5 f), indicating myelinated nerve fibers are formed followingtransplantation. Size bars 0.2 μm in FIG. 5 d-e, and 0.1 μm in FIG. 5 f.FIG. 5 g depicts bar graphs showing, as indicated, measurements of theextent of MBP stain (by color-specific integrated optical density) andmean length of MBP⁺ fibers, in similar transplantation of cells treated(red), or not treated (blue), by noggin (p<0.004 for both).

FIGS. 6 a-b depict neurons differentiated from Human ES cells which werestained for tubulin-βIII (red). Cells were cultured (as in Example 5)without (FIG. 6 a) or with (FIG. 6 b) Sonic Hedgehog (Shh) factor andwith noggin. Size bar 100 μm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of deriving neural cells, such asoligodendrocytes from human stem cells, and the use of same in treatingmedical conditions of the CNS.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The main function of oligodendrocytes is the myelination of nerve cells.The myelin sheath serves as insulation, resulting in decreased ionleakage, lower capacitance of the cell membrane and as a result allowssalutatory nerve conduction (i.e. the fast propagation ofneuroelectrical impulses). The myelin sheaths and several of its proteincomponents play essential roles in protection of neurons againstdisintegration and death. Diseases that result in injury ofoligodendroglial cells include demyelinating diseases such as multiplesclerosis, infectious or ischemic demyelinating diseases and varioustypes of inherited or acquired leukodystrophies. Information aboutfunctions of myelin and diseases caused by alterations of myelin can befound in Lazzarini et al, eds (2004) Myelin biology and disorders,Elsevier Academic Press, San Diego, Calif.).

In the past few years, research on stem cells has expanded greatly as atool to develop potential therapies to treat incurable neurodegenerativediseases.

Previous attempts to use human embryonic stem cell (huESC) lines forderiving oligodendrocytes have yielded only partial success. A firststudy described the preparation of neural tube-like rosettes which wereexpanded by bFGF into neurospheres and eventually transplanted into thethird ventricule of newborn mice brain (Zhang et al., 2001). However,only elongated bipolar O4⁺ OPC were obtained and no myelination wasshown following transplantation.

Another study (Reubinoff et al., 2001) produced neurospheres from huESCwith EGF and bFGF, and showed multipolar O4 cells with a few processes,as well as CNP-positive cells after transplantation but without evidencefor myelination. A further study, with huESC-derived neurospheres thatwere produced with bFGF and noggin yielded O4⁺ precursors but noramified or mature cells, and no transplantation was done (Itsykson etal., 2005).

In yet another study, retinoic acid was used to induce neuraldifferentiation and neurosphere production from murine ES cells (Bain etal., 1995; Liu et al., 2000). Following this approach (Nistor et al.,2005), retinoic acid was used for the induction of neural-lineage cellsfrom huESC, in concurrence with preferential selection ofoligodendrocyte-lineage cells by media components and matrigeladherence. Although “high purity functional oligodendrocytes from huESC”were claimed, no well differentiated, highly branched and ramifiedoligodendrocytes were produced, and after transplantation to spinal cordof shiverer mouse, there were only small patches of MBP-positive cellswith no evidence for extended areas of remyelination and no myelinatednerve fibers of at least 100 μm.

All the described attempts to produce fully differentiated and matureoligodendrocytes from human ES cells convey that procedures which havebeen successful with murine ES cells cannot be extrapolated to human EScells. Thus, the need for ex vivo or in vivo production of fullydifferentiated, and mature oligodendrocytes propagated and expanded fromhuman ES cells is still unmet.

While reducing the present invention to practice, the present inventorshave uncovered that incubation of human stem cells with retinoic acidand followed by incubation with noggin can be used for derivingmyelinating oligodendrocytes. Such cells can be used with greatconvenience and accessibility in the treatment of medical conditionsassociated with demyelination.

As is described herein below and in the Examples section which follows,the present inventors have uncovered, through laborious experimentation,a novel procedure for generating oligodendrocytes as well as neuronsfrom human embryonic stem cells. The general guidelines of the methodsof the present invention are further provided in Examples 1-3 and 5 ofthe Examples section which follows.

Briefly, incubation of ES cell aggregate suspension culture in thepresence of retinoic acid causes the formation of neurospheres. Spheresare allowed to ripen and outgrow on an adherent substrate. Spheres aredissociated and cells are allowed to expand through several passages inthe presence of Noggin (with bFGF and EGF at the indicatedconcentrations). Differentiation in the presence of Noggin after removalof growth factors elicits the generation of oligodendrocytes (mature aswell as expandable precursors). Such cells may be in vitro passaged andexpanded while retaining their ability to myelinate in vitro and invivo.

Thus, it is the combination of RA treatment followed by addition ofnoggin that is essential for formation of mature oligodendrocytes, whichproduce myelin proteins such as Myelin Basic Protein (MBP). In fact thepresent inventors have uncovered that noggin acts to counteract BoneMorphogenic Proteins (BMPs), formed in response to retinoic acid andwhich prevents the full differentiation oligodendrocytes.

Oligodendrocyte precursors generated according to the teachings of thepresent invention, were transplanted to shiverer mouse brains (in whichan extensive deletion in the MBP gene exists, preventing any MBPformation). Immunostaining for MBP showed that the transplanted cells ofthe present invention have the capacity to myelinate endogenous neuronsin vivo (see Example 4). Electron microscopy demonstrates the generationof thick sheaths of compact myelin with major dense lines contrastingwith the dysmyelinated nerves seen in the non-transplanted shiverermouse brain. These results substantiate the use of the cells of thepresent invention as reparative medicine of various medical conditionsof the CNS and in particular of demyelinating disease.

Thus, according to one aspect of the present invention there is provideda method of generating neuronal and glial-like cells.

The phrase “glial cells”, also termed interchangeably herein as “glia”,are non-neuronal cells that provide support and nutrition, maintainhomeostasis, form myelin, and participate in signal transmission in thenervous system. Examples of glial cells of the present invention includebut are not limited to astrocytes and oligodendrocytes (mature andprecursor, as further described herein below).

As used herein the term “oligodendrocyte” refers to both oligodendrocyteprecursor cells (OPCs) and mature well-differentiated oligodendrocytes.The function of these cells is described above. Mature oligodendrocytesmay be distinguished from OPCs both by structural and functionalphenotypes.

Examples of a mature oligodendrocyte functional phenotype include, butare not limited to one or more, marker expression such as proteolipidprotein (PLP) and MBP expression, myelin-associated glycoprotein (MAG),myelin oligodendrocyte glycoprotein (MOG), in addition togalactocerebrosides (O1, GalC).

Examples of mature oligodendrocyte structural phenotype include, but arenot limited to, a branched and ramified phenotype and formation ofmyelin membranes.

Examples of an OPC functional phenotype include, but are not limited to,mitotic (i.e. that can divide and be expanded for three or more passagesin culture) and migratory capacities as well as the potential todifferentiate into a myelinating phenotype to effect myelination in vivoand in vitro.

Examples of OPC marker expression include, but are not limited to,PDGF-receptor, O4 sulfatide marker, Nkx2.2, Sox10, Olig1/2,oligodendrocyte specific protein (OSP), 2′,3′-cyclicnucleotide-3′-phosphodiesterase (CNP), adenomatous polyposis coli (APC);NG2 (Chondroitin sulfate proteoglycan), A2B5, GD3 (ganglioside), nestin,vimentin and E- or PSA-NCAM.

Examples of OPC structural phenotype include, but are not limited toelongated, bipolar or multipolar morphology. For example only OPCs, butnot mature oligodendrocytes and astrocytes, incorporatebromodeoxyuridine (BUdR), a hallmark of mitosis.

As used herein the term “astrocytes” also termed astroglia refers to thecells which anchor neurons to their blood supply. Generally, astrocytesregulate the external chemical environment of neurons by removing excessions, notably potassium, and recycling neurotransmitters released duringsynaptic transmission. Astrocytes may be the predominant “buildingblocks” of the blood-brain barrier. Astrocytes may regulatevasoconstriction and vasodilation by producing substances such asarachidonic acid, whose metabolites are vasoactive.

Astrocytes of the present invention refer to both protoplasmic andfibrous astrocytes. Protoplasmic astrocytes have short, thick, highlybranched processes and are typically found in gray matter. Fibrousastrocytes have long, thin, less branched processes and are morecommonly found in white matter.

Astrocytes of the present invention are characterized by expression ofone or more marker, glial fibrillary acidic protein (GFAP), S100 beta,glutamine sythetase, GLAST or GLT1 and have at least one astrocyticphenotype selected from a structural astrocytic phenotypes and afunctional astrocytic phenotype. Thus, astrocytic structural phenotypesinclude a round nucleus, a “star shaped” body and many long processesthat end as vascular foot plates on the small blood vessels of the CNS.Further examples of structural astrocytic phenotypes may be found in thefollowing materials: Reynolds and Weiss, Science (1992) 255:1707-1710;Reynolds, Tetzlaff, and Weiss, J. Neurosci (1992) 12:4565-4574; andKandel, et al., Principles of Neuroscience, third ed. (1991), Appleton &Lange, Norwalk, Conn. These structural phenotypes may be analyzed usingmicroscopic techniques (e.g. scanning electro microscopy). Antibodies ordyes may be used to highlight distinguishing features in order to aid inthe analysis. Other glial cells which may be generated in accordancewith the teachings of the present invention include, but are not limitedto, olfactory-type glia and myelinating or non-myelinating Schwanncells.

As used herein the phrase “neuronal cells” refers to the polar cells ofthe vertebrate nerve system which are specialized for the transmissionof nerve impulses. Such cells typically display neuronal cell structureand express at least one neuronal marker. Examples of such markersinclude, but are not limited to neuronal and dopaminergic markersexamples of which include, but are not limited to, Peripherin, CholineAcetyltransferase [ChAT], Chromogranin A, DARPP-32, GAD65, GAD67, GAP43,HuC, HuD, Alpha internexin, MAP5, MAP-2 A&B, Nestin, NeuN, NeurofilamentL, M, H, Neuron-Specific Enolase (gamma-NSE), P75, low affinity NGFreceptor, Peripherin, PH8, Protein Gene Product 9.5 (PGP9.5), SerotoninTransporter (SERT), Synapsin, Tau, Thy-1, TrkA, Tryptophan Hydroxylase(TRH) Beta III Tubulin, TUC-4 (TOAD/Ulip/CRMP) Tyrosine hydroxylase (TH)Vanilloid Receptor Like Protein 1 (VRL-1), Vesicular GABA Transporter(VGAT) Vesicular Glutamate Transporter 1 (VGLUT1; BNPI) and VGLUT2 (allavailable at Chemicon/Millipore, Temecula, Calif.).

Oligodendrocytes, neurons and other glial cells of this aspect of thepresent invention are generated by growing human stem cells underconditions which induce the differentiation of the human stem cells intothe neuronal and glial cells. These conditions comprise the presence ofretinoic acid and an agent capable of down-regulating Bone MorphogenicProtein (BMP) activity.

As used herein the phrase “stem cells”” refers to cells which arecapable of differentiating into other cell types (i.e., neuronal orglial cells as described herein) having a particular, specializedfunction (i.e., “fully differentiated” cells) or self-renew whileremaining in an undifferentiated state. Examples of stem cells which canbe used in accordance with this aspect of the present invention include,but are not limited to, embryonic stem cells as well as fetal or adultstem cells (e.g., mesenchymal). According to currently known preferredembodiment of the present invention, the stem cells are embryonic stemcells.

It will be appreciated that undifferentiated stem cells are of adistinct morphology, which is clearly distinguishable fromdifferentiated cells of embryo or adult origin by the skilled in theart. Typically, undifferentiated stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernible cell junctions. Additional features ofundifferentiated stem cells are further described hereinunder.

The stem cells can be obtained using well-known cell-culture methods.

For example, human embryonic stem cells can be isolated from humanblastocysts or delayed blastocyst stage (as described in WO2006/040763).Human blastocysts are typically obtained from human in vivopreimplantation embryos or from in vitro fertilized (IVF) embryos.Alternatively, a single cell human embryo can be expanded to theblastocyst stage. For the isolation of human ES cells the zona pellucidais removed from the blastocyst and the inner cell mass (ICM) is isolatedby immunosurgery, in which the trophectoderm cells are lysed and removedfrom the intact ICM by gentle pipetting. The ICM is then plated in atissue culture flask containing the appropriate medium which enables itsoutgrowth. Following 9 to 15 days, the ICM derived outgrowth isdissociated into clumps either by a mechanical dissociation or by anenzymatic degradation and the cells are then re-plated on a fresh tissueculture medium. Colonies demonstrating undifferentiated morphology areindividually selected by micropipette, mechanically dissociated intoclumps, and re-plated. Resulting ES cells are then routinely split every1-2 weeks. For further details on methods of preparation human ES cellssee Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998;Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; Gardner et al.,[Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can bealso be used with this aspect of the present invention. Human ES cellscan be purchased from the NIH human embryonic stem cells registry(<http://escr.nih.gov>). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, SA01, TE03 (I3), TE04,TE06 (I6), HES-1, HES-2, HES-3, UC01, UC06, WA01, WA07 and WA09 (seealso Example 1 of the Examples section which follows).

Stem cells used by the present invention can be also derived from humanembryonic germ (EG) cells. Human EG cells are prepared from theprimordial germ cells obtained from human fetuses of about 8-11 weeks ofgestation using laboratory techniques known to anyone skilled in thearts. The genital ridges are dissociated and cut into small chunks whichare thereafter disaggregated into cells by mechanical dissociation. TheEG cells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci.USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

As mentioned hereinabove, the human stem cells of the present inventionmay be also derived from a fetal or an adult source, such as forexample, mesenchymal stem cells.

The phrase “mesenchymal stem cell” or “MSC” is used interchangeably forfetal or adult cells which are not terminally differentiated, which candivide to yield cells that are either stem cells, or which, irreversiblydifferentiate to give rise to cells of a mesenchymal cell lineage.

The mesenchymal stem cells of the present invention may be of asyngeneic or allogeneic source.

Mesenchymal stem cells may be isolated from various tissues including,but not limited to, bone marrow, peripheral blood, blood, placenta andadipose tissue. A method of isolating mesenchymal stem cells fromperipheral blood is described by Kassis et al [Bone Marrow Transplant.May 2006;37(10):967-76]. A method of isolating mesenchymal stem cellsfrom placental tissue is described by Zhang et al [Chinese MedicalJournal, 2004, 117 (6):882-887]. Methods of isolating and culturingadipose tissue, placental and cord blood mesenchymal stem cells aredescribed by Kern et al [Stem Cells, 2006;24:1294-1301].

Bone marrow can be isolated from the iliac crest of an individual byaspiration. Low-density BM mononuclear cells (BMMNC) may be separated bya FICOL-PAGUE density gradient. In order to obtain mesenchymal stemcells, a cell population comprising the mesenchymal stem cells (e.g.BMMNC) may be cultured in a proliferating medium capable of maintainingand/or expanding the cells. According to one embodiment the populationsare plated on polystyrene plastic surfaces (e.g. in a flask) andmesenchymal stem cells are isolated by removing non-adherent cells.Alternatively mesenchymal stem cell may be isolated by FACS usingmesenchymal stem cell markers. Examples of mesenchymal stem cell surfacemarkers include but are not limited to CD105+, CD29+, CD44+, CD90+,CD34−, CD45−, CD19−, CD5−, CD20−, CD11B-− and FMC7−. Other mesenchymalstem cell markers include but are not limited to tyrosine hydroxylase,nestin and H-NF.

Neural stem cells can also be used in accordance with the presentinvention. Makers such as Sox1, Sox2, SSEA-1/LeX can be used as possiblemarkers for neural cell selection. Fine separation techniques based onnegative FACS assay (in order to exclude lineage-restricted cells) andimmunomagnetic beads may be used to obtain purified and homogeneous stempopulations (Cai 2003 Blood Cells Mol Dis 31:18-27).

Regardless of their origin, stem cells used in accordance with thepresent invention are at least 50% purified, more preferably at least75% purified and even more preferably at least 90% purified. When humanembryonic stem cell lines are used, the human ES cell colonies areseparated from their feeder layer (x-ray irradiated fibroblast-likecells) such as by mechanical and/or enzymatic means to providesubstantially pure stem cell populations.

Once human stem cells are obtained, they may be treated to formaggregates as exemplified in Example 1 of the Examples section.

Cells or preferably aggregates thereof are then subjected todifferentiation conditions which comprise retinoic acids, such thatneurospheres are allowed to form. The culture medium used is selectedaccording to the stem cell used. Thus, for example, a medium suitablefor ES cell growth, can be for example DMEM/F12 (Sigma-Aldrich, St.Lewis, Mo.) or alpha MEM medium (Life Technologies Inc., Rockville, Md.,USA), supplemented with supporting enzymes and hormones. These enzymescan be for example insulin (ActRapid; Novo Nordisk, Bagsværd, DENMARK),progesterone and/or Apo transferring (Biological Industries, BeitHaemek, Israel). Other ingredients are listed in Example 1 of theExamples section.

As used herein the phrase “retinoic acid” refers to an active form(synthetic or natural) of vitamin A, capable of inducing neural celldifferentiation. Examples of retinoic acid forms which can be used inaccordance with the present invention include, but are not limited to,retinoic acid, retinol, retinal, 11-cis-retinal, all-trans retinoicacid, 13-cis retinoic acid and 9-cis-retinoic acid (all available atSigma-Aldrich, St. Lewis, Mo.).

Retinoic acid is preferably provided at a concentration range of 1-50μM.

The culture medium may be further supplemented with growth factors whichmay be present at least in part of the culturing period to promote cellproliferation and facilitate differentiation into the neuronal gliallineages. According to a preferred embodiment of this aspect of thepresent invention such growth factors include for example EGF (10-40ng/ml) and bFGF (10-40 ng/ml) (R&D Systems, Minneapolis, Minn., Biotest,Dreieich, Germany).

As used herein the phrase “neurospheres” refers to quasi-sphericalclusters or spheres containing mainly neural stem cells and earlymultipotent progenitors that can differentiate into neurons,oligodendrocytes and astrocytes as well as other glial cells.

The cells are cultured until ripened neurospheres are formed.

As used herein the phrase “ripened neurospheres” refers to neurospheresin which some of the neural stem cells have differentiated to becomespecialized oligodendrocyte progenitors having acquired makers of theoligodendrocyte lineage (e.g. Sox10, Nkx2.2, NG2, A2B5), while othershave differentiated to become neural progenitors or astrocytesprogenitors.

According to a preferred embodiment of this aspect of the presentinvention the cells are allowed to culture for example for 10-30 (e.g.,20-30) days, at the end of which detached neurospheres are formed. Thespheres, or cells dissociated therefrom, are then adhered to substratesand subjected to further expansion with growth factors and eventually todifferentiation after removal of growth factors. Examples of adherentsubstrates which can be used in accordance with the teachings of thepresent invention include, but are not limited to matrigel or anextracellular matrix component (e.g., collagen, laminin andfibronectin).

Following neuroglial sphere formation (or concomitantly with) orfollowing culture on adherent substrate, the cells are incubated with anagent capable of inhibiting Bone Morphogenetic Protein (BMP) activity.

Bone morphogenetic proteins (BMPs) are signaling molecules, belonging tothe TGF-β superfamily, which act locally on target cells to affect cellsurvival, proliferation, and differentiation, and among other actions,regulate neural cell development.

Bone morphogenetic proteins (BMPs), were shown to inhibitoligodendrocyte development from rat fetal brain (Mehler et al., 1997;Mehler et al., 2000). The present inventors have found, as illustratedin FIG. 1 c and Example 1 of the Examples section which follows, thatthe expression of several members of the BMP family, seen inundifferentiated human ES cells, increased or was even induced afterculturing cells with retinoic acid. Expression of some BMPs continued toincrease throughout the culturing steps. Since BMPs inhibitoligodendrocyte differentiation, the inhibition of these proteins iscritical for inducing differentiation of that lineage.

As used herein the term “agent capable of down-regulating BMP activity”refers to an agent which can at least partially reduce the function(i.e., activity and/or expression) of BMP.

BMP reducing agents include cystine knot-containing BMP antagonists,which are divided into three subfamilies, based on the size of thecystine ring; CAN (eight-membered ring), twisted gastrulation(nine-membered ring), and chordin and noggin (10-membered ring). The CANfamily is divided further based on a conserved arrangement of additionalcysteine residues, and includes gremlin and PRDC; cerberus and coco;DAN; USAG-1 and sclerostin.

Other BMP inhibitors include, but are not limited to, chordin like BMPinhibitor (CHL2), Neuralin (also homologous to chordin) which behave assecreted BMP-binding inhibitors; inhibin (belongs to the TGF-βsuperfamily); follistatin (which binds to inhibin); GDF3, an inhibitorof its own subfamily (TGF-β), which blocks classic BMP signaling inmultiple contexts; Crossveinless-2 (hCV-2), a BMP function inhibitor;Ectodin (available at Qiagene, Valencia, Calif.), which is homologous tosclerostin and inhibits the activity of BMP2, BMP4, BMP6, and BMP7;connective tissue growth factor (CTGF), a BMP receptor antagonist;BMP-3, a BMP receptor antagonist; and gp130 signaling cytokines.

It will be appreciated that oligonucleotide inhibitors, whichdown-regulate expression of BMP genes (e.g., siRNA) may also be used inaccordance with this aspect of the present invention. Methods ofgenetically modifying stem cells are well known in the art.

In one preferred embodiment of this aspect of the present invention theagent used to inhibit BMP is noggin (e.g., 10-100 ng/ml, e.g., 50 ng/mlused as a Noggin-Fc chimera available from R&D systems, Minneapolis,Minn.). As mentioned, noggin can be used in any culturing stepfollowing, and optionally with retinoic acid treatment.

The time and period of treatment with the BMP inhibitor followingneurospheres formation affects the differentiation level of the cells.Thus for example, a culturing period starting after the NS cells havebeen attached to adherent substrates and are cultured with growthfactors for 6-15 days and then one day without growth factors producesoligodendrocyte precursors.

A similar culture after 6 days in the absence of growth factors, but inthe presence of noggin, produces well differentiated ramified immatureoligodendrocytes.

Culturing under the same conditions for 10 days with noggin producesmature oligodendrocytes, expressing MBP.

Instead, addition of noggin early during the ripening of NS increasesdifferentiation to oligodendrocytes albeit in less magnitude thanaddition at the final culturing stage.

As is further illustrated hereinbelow and in the Examples section whichfollows, the present invention provides conditions which enable, for thefirst time, expansion of neurospheres, while retaining their functionalphenotype.

Thus, in another embodiment, the present invention provides conditionsthat enable the expansion of neurospheres for the purpose of, forexample, producing glial cells, which retain functional properties.Thus, for example, oligodendrocyte precursors generated according to theteachings of the present invention may be expanded in culture whileretaining cell capacity to migrate into affected region in the CNS, andretaining the ability to differentiate into mature oligodendrocytes.

In order to obtain populations of oligodendrocyte precursors that couldbe passaged and expanded before terminal differentiation, NS are platedon an adherent substrate and thereafter are subjected to one passage byfor example, trypsinization to yield dissociated neuroglial sphere cells(huEs-NSc). These, can be further plated on cationic substrates forfurther culture and passaging, and then be subjected to terminaldifferentiation.

Examples of adherent substrates which can be used in accordance with theteachings of the present invention include, but are not limited to,cationic substrate which can be poly-D-lysine or Polyornithine withfibronectin (FN).

As shown in the Examples section which follows, cells can be split every8-10 days for more than 3 passages, preferably more than 5 passages.

As indicated hereinabove, a BMP inhibiting agent can be added at any ofthe passages, but, as shown in Table 3 and in Example 2 of the Examplessection, inventors have found that addition of noggin at the terminaldifferentiation step (removal of growth factors), yields the highestdifferentiation into oligodendrocytes.

The present invention further provides methods for producing astrocytesfrom stem cells cultured in the presence of retinoic acid. This is doneby culturing these cells in the presence of growth factors, a BMPinhibitor (as described above), and thereafter removing the growthfactors until cells show an astrocytic phenotype (for example, culturewith noggin, without growth factors for 5-15 days).

While reducing the present invention to practice, the present inventorshave discovered that neurospheres (NS) cultured for 4 days on theadherent matrigel substrate formed a network of neurons, but thesetended to disappear after prolonged culture on matrigel. Therefore, whenculturing neurospheres for the purpose of producing neurons, inventorshave found that culturing and passaging cells on an adherent substancewhich is not matrigel or the like, does not reduce neuron formation asobserved for cells grown on matrigel.

Thus, to generate neurons, ripened NS are preferably plated on acationic adherent substance and cultured with growth factors. Inaddition to BMP cultured cells can be passaged for more than 5 times,with the addition of growth factors in every passage.

Sonic Hedgehog (Shh), a factor made in the ventral spinal cord and playsan important role in inducing defined types of neurons as well asoligodendrocyte precursors may be used to promote neuronal celldifferentiation. As described in FIG. 6 b and in Example 5 in theExamples section inventors have discovered that when Shh was addedtogether with retinoic acid an increase in the density of neuron networkwas obtained Thus, according to another preferred embodiment of thepresent invention, Sonic Hedgehog (Shh; R&D Systems, Minneapolis, Minn.)can be added together with RA.

In any of the above protocols laminin and vitamin C may be added to theculture, laminin being an extracellular matrix component which helpscells to adhere, and Vitamin C (ascorbic acid) being a well-knownantioxidant.

Populations of cells generated according to the teachings of the presentinvention may comprise for example at least about 40% of matureoligodendrocytes (comprising at least one mature oligodendrocytephenotype as described above) and any where between 0-60% cells whichcomprise a stem cell phenotype. Populations of oligodendrocyteprecursors (OPC) may comprise at least 90% of bipolar O4⁺ cells (andabout 10% of cells having a stem cell phenotype).

According to one embodiment of the present invention, the phenotype ofany of the neural or glial cells of the populations of the presentinvention is as close as possible to native cells.

Thus, as illustrated in the Examples section below, the cellsdifferentiated according to the methods of the present inventionrepresent a mature oligodendrocyte like shape, or oligodendrocyte likeprecursor shape and are accompanied by the presence of the appropriateoligodendrocyte marker.

The percentage of the cells of interest may be raised or loweredaccording to the intended needs. This may be effected by FACS using anantibody specific for a cell marker. Examples of such markers aredescribed hereinabove. If the cell marker is an internal marker,preferably the FACS analysis comprises antibodies or fragments thereofwhich may easily penetrate a cell and may easily be washed out of thecell following detection. The FACS process may be repeated a number oftimes using the same or different markers depending on the degree ofenrichment and the cell phenotype required as the end product.

Once differentiated and optionally isolated, the cells may be tested (inculture) for their phenotype. The cultures may be comparatively analyzedfor a phenotype of interest (e.g., myelin production, expansion,migration), either in vitro and/or in vivo using biochemical analyticalmethods such as immunostaining, cell expansion assays (e.g., MTT),migration assays, Western blot and Real-time PCR (some assays aredescribed in Examples 1-5 of the Examples section which follows).

Cells of the present invention may be further cloned and cell-lines ofinterest may be generated.

Cells generated according to the teachings of the present invention(described hereinabove) and in the Examples section which follows may beused in a myriad of clinical and research applications.

Thus, according to another aspect of the present invention there isprovided a method of treating a medical condition of the CNS in asubject-in-need-thereof. The method comprising administering to thesubject a therapeutically effective amount of the cells of the presentinvention (according to the intended use, as further describedhereinbelow), thereby treating the medical condition of the CNS.

Subjects treated in accordance with the teachings of the presentinvention are preferably human subjects.

As used herein, the phrase “medical condition of the CNS” refers to anydisorder, disease or condition of the central nervous system which maybe treated with the cells of the present invention.

Accordingly, these cells can be used for preparing a medicament(interchangeably referred to as pharmaceutical composition), wherebysuch a medicament is formulated for treating a medical condition of theCNS.

Representative examples of CNS diseases or disorders that can bebeneficially treated with the cells described herein include, but arenot limited to, a pain disorder, a motion disorder, a dissociativedisorder, a mood disorder, an affective disorder, a neurodegenerativedisease or disorder, an injury, a trauma and a convulsive disorder.

The cells (clones, uncloned cells of s specific type such asoligodendrocytes, or a mixed population of a number of cell types suchas oligodendrocytes and astrocytes) used may be selected according tothe intended use.

For example, the cells may comprise oligodendrocyte cells and themedical condition can be selected from, for example, the group ofautoimmune diseases, multiple sclerosis, Guillan-Barre syndrome orcongenital leukodystrophies, adrenoleukodystrophies,Pelizaeus-Merzbacher, Charcot-Marie-Tooth, Krabbe or Alexander disease,vanishing white matter syndrome, progressive multifocalleukoencephalopathy, infectious demyelinating diseases, postinflammatorydemyelinated lesions, neurodegenerative diseases, multisystemdegeneration, vascular diseases, ischemic white matter damage, vascularleukoencephalopathies, subcortical infarcts, brain trauma, spinal cordtrauma, demyelinative injury, neoplasms and oligodendrio-glioma.

In this respect, it should be noted that oligodendrocyte precursor cells(OPCs) may be adventitiously used over mature oligodendrocytes as it isprobably the mitotic and migratory capacity of these cells (in contrastto mature cells) which are vital prerequisites for successfulremyelination. According to the present invention the remyelinatingcapacity of the OPCs is significantly enhanced when the OPC have beentreated with noggin under the described conditions.

Mature differentiated oligodendrocytes may still be useful asmyelinating cells in vivo (Duncan et al. 1992 Dev. Neurosci.14:114-122). Differentiated and mature human oligodendrocytes may haveimportant applications for testing drugs that can protectoligodendrocytes from toxic or other pathogenic injuries as furtherdescribed hereinbelow.

Alternatively, the cells may comprise neurons and the medical conditioncan be selected from, for example, the group of motor neuron diseases,progressive muscular atrophy (PMA), spinal muscular atrophy (SMA),progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis,amyotrophic lateral sclerosis (ALS), neurological consequences of AIDS,Alzheimer's disease, developmental disorders, epilepsy, multiplesclerosis, neurogenetic disorders, Parkinson's disease,neurodegenerative disorders, stroke, spinal cord injury and traumaticbrain injury.

Yet alternatively, the cells may comprise astrocytes and the medicalcondition can be selected from, for example, the group consisting ofAlexander disease, epilepsy, Alzheimer's disease, spinal cord injury,traumatic brain injury and neurogenesis deficiencies.

As mentioned above, a combination of cells may be used, again dependingon the intended need. For example, a combination of oligodendrocytes andastrocytes was indicated adventitious in remyelinating a demyelinatedadult rat spinal cord (see Talbott 2005 Exp. Neurol. 11-14).

The cells of the present invention can be administered to the treatedsubject using a variety of transplantation approaches, the nature ofwhich depends on the site of implantation.

The term or phrase “transplantation”, “cell replacement” “cell therapy”or “grafting” are used interchangeably herein and refer to theintroduction of the cells of the present invention to target tissue. Thecells can be derived from the recipient or from an allogeneic orxenogeneic donor, or from embryonic stem cells.

For example, the cells can be grafted into the central nervous system orinto the ventricular cavities or subdurally onto the surface of a hostbrain or in the spinal cord. Conditions for successful transplantationinclude: (i) viability of the implant and sufficient number of cells;(ii) retention (e.g., astrocytes) or migration (e.g., OPCs) of the cellswithin the nervous tissue to the lesions in accordance with the selectedpopulation of cells; and (iii) minimum amount of pathological reaction.Methods for transplanting various nerve tissues, for example embryonicbrain tissue, into host brains have been described in: “Neural graftingin the mammalian CNS”, Bjorklund and Stenevi, eds. (1985); Freed et al.,2001; Olanow et al., 2003). These procedures include intraparenchymaltransplantation, i.e. within the host brain (as compared to outside thebrain or extraparenchymal transplantation) achieved by injection ordeposition of tissue within the host brain so as to be opposed to thebrain parenchyma at the time of transplantation.

Intraparenchymal transplantation can be effected using two approaches:(i) injection of cells into the host brain parenchyma or (ii) preparinga cavity by surgical means to expose the host brain parenchyma and thendepositing the graft into the cavity. Both methods provide parenchymaldeposition between the graft and host brain tissue at the time ofgrafting, and both facilitate anatomical integration between the graftand host brain tissue. This is of importance if it is required that thegraft becomes an integral part of the host brain and survives for thelife of the host.

Alternatively, the graft may be placed in a ventricle, e.g. a cerebralventricle or subdurally, i.e. on the surface of the host brain where itis separated from the host brain parenchyma by the intervening pia materor arachnoid and pia mater. Grafting to the ventricle may beaccomplished by injection of the donor cells or by embedding the cellsin a substrate such as 3% collagen to form a plug of solid tissue whichmay then be implanted into the ventricle to prevent dislocation of thegraft. For subdural grafting, the cells may be injected around thesurface of the brain after making a slit in the dura. Injections intoselected regions of the host brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.The microsyringe is preferably mounted in a stereotaxic frame and threedimensional stereotaxic coordinates are selected for placing the needleinto the desired location of the brain or spinal cord. The cells mayalso be introduced into the putamen, nucleus basalis, hippocampuscortex, striatum, substantia nigra or caudate regions of the brain, aswell as the spinal cord.

The cells may also be transplanted to a healthy region of the tissue. Insome cases the exact location of the damaged tissue area may be unknownand the cells may be inadvertently transplanted to a healthy region. Inother cases, it may be preferable to administer the cells to a healthyregion, thereby avoiding any further damage to that region. Whatever thecase, following transplantation, the cells preferably migrate to thedamaged area.

For transplanting, the cell suspension is drawn up into the syringe andadministered to anesthetized transplantation recipients. Multipleinjections may be made using this procedure.

The cellular suspension procedure thus permits grafting of the cells toany predetermined site in the brain or spinal cord, is relativelynon-traumatic, allows multiple grafting simultaneously in severaldifferent sites or the same site using the same cell suspension, andpermits mixtures of cells from different anatomical regions. Multiplegrafts may consist of a mixture of cell types, and/or a mixture oftransgenes inserted into the cells. Preferably from approximately 10⁴ toapproximately 10⁸ cells are introduced per graft.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the central nerve system (CNS) to form a transplantationcavity, for example as described by Stenevi et al. (Brain Res. 114:1-20,1976), by removing bone overlying the brain and stopping bleeding with amaterial such a gelfoam. Suction may be used to create the cavity. Thegraft is then placed in the cavity. More than one transplant may beplaced in the same cavity using injection of cells or solid tissueimplants. Preferably, the site of implantation is dictated by the CNSdisorder being treated.

For example, in treating multiple sclerosis transplantation into a smallnumber of carefully chosen lesions, for example, the optic nerves, thespinal cord, or the superior cerebellar peduncle can be effected.

In other inherited disorders of myelin metabolism, a systemic mode ofadministration may be used to exploit the migratory capacity of OPCs andboth the circulation of the brain and the blood. In these casesdisruption of the blood-brain barrier and/or supplementation with growthfactor infusion or growth/trophic factor secreting cells.

Since non-autologous cells are likely to induce an immune reaction whenadministered to the body, several approaches have been developed toreduce the likelihood of rejection of non-autologous cells. Theseinclude either suppressing the recipient immune system or encapsulatingthe non-autologous cells in immunoisolating, semipermeable membranesbefore transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesTechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Human ES cells that are “patient specific” and therefore would not besubject to immune reaction may be obtained by nuclear transfer inenucleated oocytes (so called therapeutic cloning). Alternatively, humanES cell banks may be used to find cells that are HLA matched to thepatient. In addition, genetic engineering of the ES cells may be done todown-regulate histocompatibility antigens and reduce the risk of immunereaction. For this purpose, it is possible to use siRNA or some viralgenes (Lee E M, Kim J Y, Cho B R et al, Biochem. Biophys. Res Commun.326, 825-835, 2005).

In any of the methods described herein, the cells can be administeredeither per se or, preferably as a part of a pharmaceutical compositionthat further comprises a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the chemical conjugates described herein, with otherchemical components such as pharmaceutically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to a subject.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the present invention, thepharmaceutical carrier is an aqueous solution of saline.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration include direct administration into thetissue or organ of interest. Thus, for example the cells may beadministered directly into a specific region of the brain or to thespinal cord.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Preferably, a dose is formulated in ananimal model to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. For example, animal models ofdemyelinating diseases include shiverer (shi/shi, MBP deleted) mouse, MDrats (PLP deficiency), Jimpy mouse (PLP mutation), dog shaking pup (PLPmutation), twitcher mouse (galactosylceramidase defect, as in humanKrabbe disease), trembler mouse (PMP-22 deficiency). Virus induceddemyelination model comprise use if Theiler's virus and mouse hepatitisvirus. Autoimmune EAE is a possible model for multiple sclerosis.

Animal models for neuronal diseases include 6-OHDA-lesioned mice whichmay be used as animal models of Parkinson's. In addition, a sunflowertest may be used to test improvement in delicate motor function bychallenging the animals to open sunflowers seeds during a particulartime period.

Transgenic mice may be used as a model for Huntington's disease whichcomprise increased numbers of CAG repeats have intranuclear inclusionsof huntingtin and ubiquitin in neurons of the striatum and cerebralcortex but not in the brain stem, thalamus, or spinal cord, matchingclosely the sites of neuronal cell loss in the disease.

Transgenic mice may be used as a model for ALS disease which compriseSOD-1 mutations.

The septohippocampal pathway, transected unilaterally by cutting thefimbria, mimics the cholinergic deficit of the septohippocampal pathwayloss in Alzheimers disease. Accordingly animal models comprising thislesion may be used to test the cells of the present invention fortreating Alzheimers.

The data obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition, (see e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1). For example,Parkinson's patient can be monitored symptomatically for improved motorfunctions indicating positive response to treatment.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Dosage amount and interval may be adjusted individually to levels of theactive ingredient which are sufficient to effectively regulate theneurotransmitter synthesis by the implanted cells. Dosages necessary toachieve the desired effect will depend on individual characteristics androute of administration. Detection assays can be used to determineplasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the individual being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administration willbe responsive to a careful and continuous monitoring of the individualchanging condition. For example, a treated Parkinson's patient will beadministered with an amount of cells which is sufficient to alleviatethe symptoms of the disease, based on the monitoring indications.

The cells of the present invention may be co-administered withtherapeutic agents useful in treating neurodegenerative disorders, suchas gangliosides; antibiotics, neurotransmitters, neurohormones, toxins,neurite promoting molecules; and antimetabolites and precursors ofneurotransmitter molecules such as L-DOPA. Additionally, the cells ofthe present invention may be co-administered with other cells.

Following transplantation, the cells of the present invention preferablysurvive in the diseased area for a period of time (e.g. at least 6months), such that a therapeutic effect is observed. As described inExample 4, the cells of the present invention were shown to migratemyelinate in shiverer mouse brain.

Generally, any method known in the art can be used to monitor success oftransplantation. For example, MRI can be used for visualizing brainwhite matter and studying the burden of delyelinating lesions ascurrently practiced for monitoring MS patients. Magnetization transfercontrast can be used to monitor remyelination (Deloire-Grassin 2000 J.Neurol. Sci. 178:10-16). Magnetic resonance spectroscopy measurement ofN-acetyl-aspartate levels can be used to assess impact on localneuron/axon survival. Using paramagnetic particles to label cells beforetransplantation enabling their dispersion to be tracked by MRI. Serialneurophysiology is useful for monitoring conduction. The optic nerve hasparticular advantages in this respect.

Other approaches to more generalized neurophysiological assessment aredescribed in Leocani et al. Neurol Sci. 2000;21(4 Suppl 2):S889-91 whichmay be useful for interventions aimed at multifocal or more diffusemyelin repair. Notwithstanding, it is appreciated that clinicalimprovement may also be assessed. Demyelination causes alterations ofstature (trembling, shivering) and locomotion. Children withleukodystrophies have motor and intellectual retardation.Electrophysiological measures of sensory and motor nerve conductivity,for example H-wave, are classical method used in monitoring neuropathieslinked to demyelinating peripheral lesions (Lazzarini et al, eds (2004)Myelin biology and disorders, Elsevier Academic Press, San Diego,Calif.).

As mentioned above, cells of the present invention can be used as animperative tool for in vitro screening of drugs.

Thus, according to yet another aspect of the present invention there isprovided a method of determining an effect of a treatment on neural cellfunctionality, the method comprising subjecting a cell of the presentinvention (e.g., oligodendrocyte) to the treatment (e.g., drug,condition such as electrical treatment and an irradiation treatment);and determining at least one of a structural or functional phenotype ofthe treated cell as compared to an untreated cell, thereby determiningan effect of the treatment on neural cell functionality.

Qualifying the effect of a treatment of interest on the cells of thepresent invention can be used to identify and optimize treatmentscapable of restoring the neural function, and hence can be used toidentify and optimize drugs suitable for treating neural disorders(e.g., including treatment methods envisaged by the present invention).

Furthermore, qualifying the effect of a treatment (either directed todiseases of the CNS or any other tissue) on neural functionality can beused to assess the toxicity of such clinical treatments.

Thus, this aspect of the present invention can be preferably utilized todetermine the therapeutic and toxic effects of various treatments, suchas drug treatments, and electrical treatments, on neural function.

Hence the method of the present invention can be used to screen and/ortest drugs.

This aspect of the present invention can be also utilized to obtain geneexpression profiles and changes thereof in cells of the presentinvention subjected to a treatment. Thus, the method according to thisaspect of the present invention can be used to determine, for example,gene expression pattern changes in response to a treatment.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Examples

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Conversion of Human ES Cells into Cells Expressing GenesCharacteristic of the Oligodendrocyte Lineage

ES cells were cultured with a combination of retinoic acid (RA)treatment followed later by the addition of Noggin addition to producemature oligodendrocytes, defined by the expression of myelin proteins.

Materials and Experimental Procedures

Culturing Media—

ES1: ESC growth medium—The growth medium (ES1) consisted of DMEM/F12(Sigma-Aldrich, St. Lewis, Mo.) supplemented with 20% knockout serumreplacement (KSR, Gibco BRL/Invitrogen, Gaithersburg, Md.), MEMnonessential amino acids (1/100; Gibco BRL/Invitrogen, Gaithersburg,Md.), 100 μM beta-mercaptoethanol (Gibco BRL/Invitrogen, Gaithersburg,Md.), 1 mM Na pyruvate, 2 μg/ml heparin and with 6 to 8 ng/ml humanrecombinant basic fibroblast growth factor (bFGF, Cytolab, Rehovot,Israel).

ITTSPP/B27 medium—The medium was composed of 50% (v/v) of DMEM/F12(Sigma-Aldrich, St. Lewis, Mo.) containing 2% B27 supplement (GibcoBRL/Invitrogen, Gaithersburg, Md.) and 50% (v/v) of DMEM/F12 containing25 μg/ml insulin (ActRapid; Novo Nordisk, Bagsværd, DENMARK), 50 μg/mlhuman Apo transferrin (Biological Industries, Beit Haemek, Israel), 6.3ng/ml progesterone (Sigma P01304), 10 μg/ml putrescine (Sigma-Aldrich,St. Lewis, Mo.), 50 ng/ml sodium selenite (Sigma), and 40 ng/mltriiodothyronin (T3, Sigma), with penicillin (100 U/ml) and streptomycin(100 μg/ml).

T-medium—50% (v/v) ES1 medium, and 50% (v/v) of ITTSPP/B27 medium.

N2/B27 medium—The medium was composed of DMEMIF12, 0.5% (v/v) N2supplement (Gibco BRL/Invitrogen, Gaithersburg, Md.), 1% (v/v) B27supplement, 10 ng/ml of EGF, and 10 ng/ml of bFGF

Human ESC line culturing—Human embryonic stem cell lines (huESC; forexample, I-3 or I-6 as in Amit and Itskovitz-Eldor, 2002), were cultured(in ES1), frozen and thawed, as previously described (Amit et al.,2004), on feeder layers of mouse embryonic fibroblasts (MEF) which havebeen gamma-irradiated at 37° C., in a 5% CO₂ incubator. About 4 daysfollowing seeding, huES cells colonies were washed with DMEM/F12, anddetached by collagenase IV (Worthington Biochemical Corp. Lakewood,N.J.), freshly prepared, 1.2 mg/ml in DMEM/F12, using 1 ml per well for45 to 90 minutes in the incubator. The detached aggregates werethereafter cultured as described above or subjected to thedifferentiation protocol as described hereinbelow and outlined in Table1.

Differentiation of ES cells into oligodendrocytes—The following stepswere taken for initiating ES cell differentiation:

1. Step T: Transition step—Collagenase detached huESC colonies pooledfrom six 6-well plates, into a 15 ml tube supplemented with ES1 medium,were let to sediment by gravity for about 30 minutes in the incubator(37° C.). Colonies were then washed once in ES1 medium and thentransferred to 12×6 cm tissue culture plates in 5 ml transition medium(T-medium) consisting of 50% (v/v) ES1 medium, and 50% (v/v) ofITTSPP/B27 medium. Growth factors (GF), recombinant human EGF (20 ng/ml,R&D Systems, Minneapolis, Minn.) and recombinant human bFGF (4 ng/ml,Biotest, Dreieich, Germany) were added to the transition medium.Thereafter (1 d), some cells adhered but the bulk of huESC colonies andaggregates remained in suspension and were transferred to 12 bacterial(non-adherent) plates in the same transition medium and cultured for 1day. Then, 10 μM retinoic acid (RA, Sigma-Aldrich, St. Lewis, Mo.) wasadded to the T-medium and cells were cultured for an additional day.

2. Step R: Retinoic acid treatment—The medium was switched to ITTSPP/B27containing 20 ng/ml of EGF and 10 μM RA. Medium was changed every dayfor 7 days, and colonies were concentrated by gravity, withoutcentrifugation.

3. Step S: Suspension culture—During this step, which allows forripening of neurospheres (NS), the aggregates were cultured inITTSPP/B27 medium supplemented with 20 ng/ml EGF, for 18 days, changingmedium every two days. The ITTSPP/B27 medium was, or was notsupplemented with 50 ng/ml Noggin-Fc chimera (R&D systems, Minneapolis,Minn.), in order to examine the effect of noggin. The addition of bFGF20 ng/ml at this step was not found to be helpful.

4. Steps M1,2: Adherence to matrigel-coated plates—Spheres or aggregatesfrom one original 6-well plate were distributed to another 6-well platewhich had been coated for 1.5 hours at room temperature with matrigel(Matrigel low growth factor concentration; BD Biosciences, Clontech,Palo Alto, Calif., diluted 1:30 in DMEM/F12 Pen-Strep). After one day,the unbound aggregates were discarded and fresh ITTSPP/B27 medium,supplemented with 20 ng/ml EGF, was added. Medium was changed every 3-4days.

5. Passage with trypsin—Following 1-2 weeks, aggregates were detachedand partially dissociated by a short treatment with trypsin 0.05% (2-3min , 37° C.; Biological industries, Beit Haemek, Israel, diluted inPBS, —Ca and —Mg). Trypsin was rapidly neutralized by addition of 4volumes of BSA (2 mg/ml; Sigma for Tissue culture) in ITTSPP/B27, andcentrifugation at (200 rpm, 10 min), followed by another wash with BSA.This treatment dissociates the aggregates to small clusters that areseeded again onto matrigel-coated plates (at dilution 1:3), inITTSPP/B27, 20 ng/ml EGF, for another 1-2 weeks (step M2).

6. Steps F1,2: Terminal differentiation on matrigel—In step F1 (3 days),the medium was changed to ITTSPP/B27 containing EGF 5 ng/ml and bFGF 5ng/ml, together with mouse laminin 1 μg/ml, and Vit C (50 μg/ml;Sigma-Aldrich, St. Lewis, Mo.). Some of the wells were treated withnoggin (50 ng/ml Noggin-Fc chimera; R&D systems, Minneapolis, Minn.), 2days after seeding, and noggin was continued thereafter. In step F2,cells were cultured 6 or 10 days in the same medium without growthfactor, but with or without noggin. The medium was changed every two tofour days.

The procedures and different conditions used for derivingoligodendrocytes from human ES cells described above are summarized inTable 1 below, several culturing conditions (e.g., addition of steps, ortime duration of steps) were administered to the cells (numbered 1-4 inTable 1). Conditions 2-4 are further explained in Example 2-5.

TABLE 1 Step: Conditions (conc.): 1 2 3 4 T EGF (20)#, bFGF (4) in 2 dES1/[ITTSPP/B27] R Retinoic acid 10 μM + EGF 7 d (20), [ITSPP/B27] S EGF(20) in [ITSPP/B27] 18 d ± 18 d ± 18 d 18 d noggin noggin M1 EGF (20) in[ITSPP/B27] 9 d 17 d 13 d 13 d (matrigel) M2 Trypsin passage (P1) and —11 d 21 d 21 d (matrigel) replate in same D1 Trypsin (P2) and replate in— Trypsin dissociation of the cells (dissociated EGF (20) in [ITSPP/B27]PolyornithineF Poly D- Poly D- cells) N, laminin Lysine Lysine 3 d 1 d 1d D2 EGF (10) + bFGF (10) in — 8 d ± 15 d 15 d (dissoc. cells) [N2/B27]noggin D3 Passage (P3, Hanks) and — — 6 d ± 13 d ± (dissoc. cells)replate in same noggin noggin D4 Passage (P4, Hanks) and — — — 8 d ±(dissoc. cells) replate in same noggin F1 EGF (5) + bFGF (5) in 3 d* ± 3d ± — — [N2/B27] noggin noggin F2 No GF in [N2/B27] 6 or 10 d* ± 6 d ± 6or 10 d ± 6 d ± noggin noggin noggin noggin *differentiation step Fwhile still on matrigel and in ITSPP/B27 medium #concentrations inng/ml.

RT-PCR—total RNA was isolated from cultures at different culture stepsusing Tri-Reagent (Molecular Research Center, Cincinnati, Ohio) andRT-PCR was carried out by standard technology as previously described(Slutsky et al., 2003). Expression was checked for the following genes,as described in table 2 below,including the expression of the controlgene G3PDH. Also provided in the table are primer sequences used foreach gene. For G3PDH, primers from Clontech (Palo Alto, Calif., catalog#9805-1) were used.

TABLE 2 Human Nucleotide Accession SEQ ID Gene Sequence number numberNO. BMP-7 F CCTTCATGGTGGCTTTCTTC 946-965 NM_001719  1 BMP-7 RCCAAAGGGTCTGAATTCTCG 1444-1425  2 BMP-2 F AGAACTACCAGAAACGAGTGGG1142-1163 NM_001200  3 BMP-2 R CGCTGTTTGTGTTTGGCTTG 1651-1632  4 BMP-6 FCGGTTCTTTACTTTGATGAC 1639-1658 NM_001718  5 BMP-6 RGATAGACAGTACTGAACCAGC 2142-2122  6 BMP-4 F CATTTATGAGGTTATGAAGCC1014-1033 NM_001202  7 BMP-4 R CCACCTTATCATACTCATCCAG 1654-1633  8BMP-11 F GCGACTAAAACCCCTAACTG 632-651 AF100907  9 BMP-11 RCTGCACCAAATGGGTATGC 1118-1099 10 Nkx2.2 F TGCCTCTCCTTCTGAACCTTGG1388-1409 NM_002509 11 Nkx2.2 R GCGAAATCTGCCACCAGTTG 1724-2705 12 OCT3-4F CTTGCTGCAGAAGTGGGTGGAGGAA 662-686 NM_002701 13 OCT3-4 RCTGCAGTGTGGGTTTCGGGCA 830-810 14 olig1 F TTGCATCCAGTGTTCCCGATTTAC1688-1711 NM_138983 15 olig1 R TGCCAGTTAAATTCGGCTACTACC 2077-2054 16olig2 F AAGGAGGCAGTGGCTTCAAGTC 368-388 NM_005806 17 olig2 RCGCTCACCAGTCGCTTCATC 681-662 18 PDGFR F CTATCCACACTGTCAAACAGGTTG5299-5322 NM_006206 19 PDGFR R TCTGCTGGACTGAGAAGTTTCATC 5751-5729 20 PLPF CTGCTCACCTTCATGATTGC 825-844 NM_000533 21 PLP R TGACTTGCAGTTGGGAAGTC1149-1168 22 REX-1F TGAAAGCCCACATCCTAACG 1283-1302 NM_174900 23 REX-1RCAAGCTATCCTCCTGCTTTGG 1839-1819 24 SOX 10 F GCCTGTTCTCCTGGGGGTTTGCTGC1889-1865 NM_006941 25 SOX 10 R CATGCACCTCACAGATCGCCTACAC 1396-1420 26TGFβ1 F CAGCAACAATTCCTGGCGATAC 1389-1410 NM_000660 27 TGFβ1 RGGACAGCTGCTCCACCTTG 2007-1989 28

Results

In order to examine gene expression during the different culture steps,and the effect of noggin on this expression, RT-PCR was effected forgenes expressed during ES maturation to oligodendrocytes, in thedifferent culture steps, with or without the addition of noggin. FIG. 1a shows the expression of genes for the transcription factors Nkx2.2,Sox10 and Olig2 believed to be characteristic of the oligodendrocytelineage (Gokhan et al., 2005). Whereas Olig1 and Olig2 were expressedafter step R (FIG. 1 a, lane 1; retinoic acid treatment), the expressionof the two other genes was seen only when noggin was added, after step S(Suspension of culture, where neurospheres are allowed to ripen; FIG. 1a, lane 3). Sox10 was not seen after step S without noggin (FIG. 1 a,lane 2). FIG. 1 b shows that using a protocol which was successful fordifferentiation of murine ES cells into oligodendrocytes (Zhang et al.,2004; Zhang et al., 2006), based on bFGF treatment but with neither RAnor noggin, failed to induce Nkx2.2 in human ES cells, while Sox10 (andOlig2, not shown) were expressed. The same was observed when 40 ng/mltriiodothyonine was added (FIG. 1 b, right lane). In addition,expression of growth factor receptor PDGF-Rα, characteristic of earlyprecursors of the human oligodendrocyte lineage (Zhang et al., 2000) washigh following RA treatment (FIG. 1 a, lane 1) and decreased as expectedlater (FIG. 1 a, lane 2) and more so if noggin was added (FIG. 1 a, lane3). This is an indication that noggin acts on differentiation of OPcells. The myelin proteolipid protein (PLP) is seen increasing, whereasmarkers of undifferentiated pluripotent stem cells, such as Oct4 andRex1, are down-regulated after step S with noggin (lane 3). Therationale for adding noggin to cultures treated with retinoic acid isillustrated by FIG. 1 c. Noggin is an inhibitor counteracting the effectof Bone morphogenetic proteins (BMPs), which were shown to inhibitoligodendrocyte development from rat fetal brain (Mehler et al., 1997;Mehler et al., 2000). FIG. 1 c shows that the expression of severalmembers of the BMP family, seen sometimes in undifferentiated human EScells (lane 1), is increased or even induced after step R (FIG. 1 c,lane 2). Expression of some BMPs, such as BMP-6 and 7, continues toincrease at step S, M and F (lanes 4, 5, 6). These BMPs are likely to bethe targets of noggin's effect.

Example 2 Effect of Noggin on the Terminal Differentiation of HumanOligodendrocytes Derived from ES Cells

Effect of noggin on human oligodendrocyte differentiation was examinedby direct visualization and counting of immunostained cells culturedwith and without noggin.

Materials and Experimental Procedures

Culture and culture conditions—culture conditions were effected asdescribed in Example 1.

Immunostaining—Cells were fixed in 12 or 24-well tissue culture plasticplates with 4% paraformaldehyde (PFA), washed with PBS, and kept at 4°C. before staining. Non-specific staining was blocked with normal goatserum (5% w/v in PBS) for 30 min at RT. Thereafter primary mousemonoclonal (mMc) antibodies, diluted in 1% goat serum, wereadministered. Antibodies used were: anti PDGFR (mMc IgG1, Santa Cruz,1:500), O4 (mMc IgM, R&D Systems, 1:1000), O1 (mMc IgM, received from DrSheila Harroch, Pasteur Institute, Paris), anti tubulin-βIII (mMc IgG1,Cowance, 1:1000), and antibody 1281 (human nuclei antigen HNA,Chemicon). Following overnight exposure to the appropriate antibody (4°C.), biotinylated goat anti-mouse IgG1 (Southern Biotech, 1:50) wasadded for 30 minutes, followed by Cy3-tagged streptavidine (ABClaboratories, 1:2000). For double staining, O4 or O1 was revealed withanti mIgM-FITC (Chemicon, 1:75, 1 hour) and cells fixed again with 3%PFA for 10 minutes, permeabilized by 0.2% Triton-X100 for 10 min whennecessary, and reacted with the other antibody. Staining for MBP waswith rat monoclonal anti MBP (ab7349, AbCam, 1/500), and Cy3-conjugatedAffipure goat anti rat IgG (Jackson laboratories, 1:500). Staining forGFAP was with Cy3-conjugated mMc anti-GFAP (Sigma-Aldrich, St. Lewis,Mo.; 1:1000). Staining was effected for 1 hour at RT, followed by thenuclear fluorescent dye DAPI (Sigma-Aldrich, St. Lewis, Mo., 0.05μg/ml). All coverslips were mounted in Mowiol (Calbiochem, LaJolla,Calif.). An Olympus IX-70 FLA microscope with a DVC-1310C digital camera(DVC, Austin, Tex.) was used and images were processed with Photoshopand analyzed with Image-ProPlus software (Media Cybernetics, SilverSpring, Md.), in order to measure Integrated Optic Density (IOD) ofcolor-specific pixels and MBP fiber length. Statistical analyses waswith two-tailed Student t-test and Anova, using Instat (GraphPadSoftware Inc.) or JMP 5.0 software (SAS Institute, Cary, N.C.)

Cell count—The number of O4⁺ cells with oligodendrocyte branching 2morphology was counted in fields of 0.4 mm². Cells were countedfollowing the differentiation step, with cells on matrigel, as in Table1, condition 1, described in Example 1. Additionally, dissociatedneurosphere cells plated on poly-D-lysine (PDL) for expansion anddifferentiation, as described in Table 1, conditions 2,3,4 respectively,were counted. In all cases, cells were fixed after 6 days ofdifferentiation without growth factors. p-value was obtained bytwo-tailed Student's t-test.

Results

Immunostaining—In order to follow cell development under theadministered culture conditions, immunostaining was done with antibodiesto neuron-specific tubulin-βIII to follow the creation of neurons, andwith antibodies against oligodendrocyte-specific marker O4⁺, theastrocyte marker GFAP and the oligodendrocyte precursor marker PDGFR, tofollow glial cell types and the developmental stage of the cells.Results show that after 4 days on matrigel (step M, as described inExample 1) the outgrowth from the neurospheres contains a network ofneural processes and a few round cells labeled by the O4⁺ marker (FIG. 1d). After 8 days in step M, the O4⁺ cells (green) are still of smallround, monopolar morphology (FIG. 1 e, panel 1 and 3) and are alsostained for the astrocyte marker GFAP (red, FIG. 1 e, panel 2). Muchless neurons survived at this stage (not shown). Upon removal of EGF andaddition of noggin for 6 days (still on matrigel, as seen in FIG. 1 f),the O4⁺ cells develop into elongated bipolar cells, staining positivefor the PDGF-receptor (red), which is a marker of oligodendrocyteprecursors. Notably, a number of O4⁺ cells have already undergoneterminal differentiation to highly branched oligodendrocytes (green),and in accordance have down-regulated the PDGF-receptor. After 10 dayswithout growth factors, a strong effect of noggin on the number ofwell-developed oligodendrocytes displaying ramified branches, is evident(FIGS. 1 g-h). Without noggin (FIG. 1 g), most O4⁺ cells are stillelongated bipolars, whereas with noggin (FIG. 1 h) many O4⁺ cells withthe typical oligodendrocyte morphology are seen. Table 3 given belowshows quantitative results of such experiments showing that the additionof noggin at step F1,2 (terminal differentiation steps, as described inTable 1, conditions 1) produces the highest increase in oligodendrocytesper field. Addition of noggin at step S (ripening of neurospheres) alsoincrease oligodendrocyte number albeit less than addition at theterminal step F.

TABLE 3 Number of oligodendrocytes per field Conditions Without NogginWith Noggin Fold p-value A. On Matrigel Add noggin at: Step F 0.66 ± 0.6(N = 15) 5.76 ± 4.2 (N = 25) 8.7 0.0001 Step S 1.50 ± 1.4 (N = 14) 2.20.043 Steps F and S 2.95 ± 1.7 (N = 20) 4.4 0.0002 B. Dissociated cellsPassage 2 0.57 ± 0.5 (N = 7) 5.20 ± 1.7 (N = 10) 9.1 0.0001 Passage 31.43 ± 3.1 (N = 16) 10.14 ± 5.2 (N = 7) 7.1 0.0001 Passage 4 2.00 ± 1.4(N = 9) 12.05 ± 3.6 (N = 6) 6.0 0.0001

O4 is a marker of immature oligodendrocytes, but staining for the MBPprotein (FIG. 2 a) shows that many of the O4⁺ cells (FIG. 2 b) expressthis myelin component indicating that they are maturatingoligodendrocytes. Some cells are double positive (see also FIG. 2 c),some are still only O4⁺ (FIG. 2 d) and some have matured to the stagewhere O4 has ceased to be expressed in the MBP-positive oligodendrocytes(FIG. 2 e). The number of MBP-expressing cells is high after addition ofnoggin (FIG. 2 f), and quantitation of the effect of noggin (FIG. 2 g)shows a very significant stimulation of the number of MBP⁺oligodendrocytes

Example 3 Dissociated Neuroglial Sphere Cells (NSc) Differentiating intoMature Oligodendrocytes

Materials Experimental Procedures

Culture and culture conditions—culture conditions were effected asdescribed in Example 1.

Cell expansion—In order to obtain populations of oligodendrocyteprecursors that could be passaged and expanded before terminaldifferentiation, the clusters, aggregates or spheres on matrigel weresubjected to one passage by mild trypsinization (as described inExample 1) while still on matrigel (passage 1), and then detached frommatrigel and dissociated by trypsin to yield dissociated cells. Thesehuman ES cell-derived dissociated neuroglial sphere cells (huEs-NSc),were plated onto cationic substrates for further culture (passage 2),and then terminal differentiation according to the following steps, asoutlined in Table 1, conditions 2-4, in Example 1.

Step D1: Dissociated cells plated on cationic substrates—Tissue cultureplates were coated with bovine plasma fibronectin (250 μg/ml FN,Sigma-Aldrich, St. Lewis, Mo.) diluted in PBS (plus Ca and Mg)transferred from well to well, dried and coated with poly-L-ornithine(PO, Sigma-Aldrich, St. Lewis, Mo.) and washed three times with water.Alternatively, tissue culture plates were coated with Poly-D-Lysine (20μg/ml PDL, Sigma-Aldrich, St. Lewis, Mo.), in 10 mM borate buffer, for 2hours to overnight at 37° C., and washed three times with sterile water.The huES-NS cells or small aggregates, detached from matrigel anddissociated by trypsin, were seeded on PDL or FN-PO coated plates inITTSPP/B27 supplemented with EGF 20 ng/ml for one day.

Steps D2-4: Expansion of dissociated huES-NSc—One day after culturingwith ITTSPP/B27 supplemented with EGF 20 ng/ml, the medium was changedto N2/B27 medium, and mouse laminin (1 μg/ml, Sigma-Aldrich, St. Lewis,Mo.) was added to the medium during the first seeding. Cells were splitevery 8 to 10 days.

For passage 3 to passage 5, the cells from PDL plates were dissociatedin Hank's balanced salt solution (HBSS; Invitrogen/Gibco, Gaithersburg,Md.) without Ca and Mg. Cells were first quickly washed with warm HBSS,and immediately added with 0.4 ml of HBSS per well, while being scrapedgently with a rubber policeman. The cells were then centrifuged,dissociated by up and down pipetting, and reseeded (at 1:2 dilution)onto PDL-coated plates in N2/B27 with laminin. Cells split with HBSShave a tendency to form rosette-type aggregates. When aggregates weremore than 0.5 mm diameter, and could not be dissociated with HBSS,0.025% trypsin-EDTA, was added for exactly 2 min, and neutralizationwith BSA. Surviving cells formed monolayers and very small aggregates,and were usually split 1:3. In order to define the effect of noggin oncell development, 50 ng/ml noggin was added at these steps, as indicatedin Table 1, Example 1.

Step F2: Terminal differentiation of dissociated cells—At the differentpassages, terminal differentiation was induced by removing the growthfactors for 6-10 days. Step F1 (as described in Example 1) could beomitted (see Table 1 conditions 3, 4). Whenever growth factors wereremoved, 1 μg/ml mouse laminin, and 50 μg/m Vit C were added to theN2/B27 medium. Noggin, 50 ng/ml, was continued as indicated in Table 1.

O1 staining—staining with O1 antibodies specific for matureoligodendrocytes was effected as previously described (Zhang et al,2004).

Results

Effect of noggin on oligodendrocyte development—in order to assess theeffect of noggin addition to oligodendrocyte development, the number ofoligodendrocytes was compared in fields examined from cells culturedwith and without noggin addition, at different culture steps, asdescribed in Examples 1 and 2 and in Table 1. Addition of noggin wasfound to increase the number of O4⁺ cells with oligodendrocytemorphology (multiple ramified branches) by 9-fold, as presented in Table3, B, Passage 2). At this passage, the total O4⁺ cells represented about15% of the total cells, and those with ramified oligodendrocyte shapewere about 5% of the total cells. Results also confirm that addition ofnoggin at the early step S, resulted in a much lower fold increase ofoligodendrocytes formed in the differentiation step (only 1.77±1.2versus 5.20±1.7, shown for passage 2 in table 3; p<0.0001). The use ofbFGF alternating with EGF during step S (without noggin) was deleteriousand only 0.66±0.5 oligodendrocytes formed in the final differentiationstep F with noggin, a much lower value than 5.20±1.7 which was obtainedif EGF alone was used in step S (see Table 3B). While this earlyexposure to bFGF impaired oligodendrocyte development, it wasnevertheless found that the combination of bFGF and EGF was better thanEGF alone for the expansion of dissociated precursor cells at step D2.Hence, the timing of factor addition was found to be very critical inthis procedure. In addition, purity and differentiation capacity ofoligodendrocytes increased after multiple passages. The sub-passagedculture (fixed within a day after passage 4) showed a rather homogeneouspopulation of O4⁺ bipolar cells (as shown in FIG. 3 a). Indeed, lessthan 10% of the nuclei belonged to O4 negative cells. The bipolar cellscould be differentiated (10 days without GF, but with noggin) to formdense fields of highly branched O4⁺ oligodendrocytes (FIG. 3 b).Differentiated cells exhibited the complex branching pattern and theformation of flat membranes, typical of maturating oligodendrocytes, asclearly seen in FIG. 3 c

To further assess Development of mature oligodendrocytes, staining withO1 antibodies specific for mature oligodendrocytes was effected, asdescribed previously (Schachner et al., 1981). O1 staining was found tobe high in the cell bodies and in the branches (FIG. 3 d, green).Astrocytes, stained for GFAP were observed as well (FIG. 3 d, red).

A higher number of oligodendrocytes per field, formed after terminaldifferentiation, was found at passages 3 and 4 than at earlier steps(Table 2, B). The stimulating effect of noggin addition during steps Dand F was clearly seen in all cases (Table 2,B). Although moreoligodendrocytes formed in the control cultures at the higher passages,the proportion of oligodendrocytes became higher after thesesub-passages and reached 43% of the total cells in the culture.

Taken together, these results show that the capacity of thehuESC-derived precursors (OP) to differentiate into oligodendrocytes isnot only conserved through several passages but actually increases,further substantiating the ability of the present invention to makeslarge scale expansion of oligodendrocytes possible.

Example 4 The Effect of Noggin on the Myelination Capacity of HumanOligodendrocyte Precursor Cells In Vivo

Myelination capacity of human oligodendrocyte precursor cells aseffected by noggin was determined in vivo in shi^(−/−) Shiverer mice,which lack MBP immunoreactivity in the CNS

Reagents and Experimental Procedures

Animal model—Shiverer mice have an extensive deletion in the MBP gene(Roach et al., 1985) and in a homozygous shi−/− animal there is no MBPimmunoreactivity in the CNS. The appearance of fibers showingimmunostaining for MBP following transplantation of oligodendrocyteprecursors therefore indicates, that the transplanted cells have thecapacity to myelinate endogenous neurons (Lachapelle et al., 1983).

Transplantation of huES-NSc in Shiverer mice—For transplantation, EScell-derived dissociated neuroglial sphere cells (huEs-NSc) wereprepared as described in Example 3. The culture steps from step T tostep D3 that were followed, are outlined in Table 1 condition 3. inExample 1. However, Step D3 was for 9 days, with and without 50 ng/mlnoggin. Growth factors were then removed for one day, during which 1μg/ml laminin, and 50 μg/m Vit C were added to the N2/B27 medium (stepF2, as described in Example 1). The culture was thereafter subjected tothe short trypsin treatment (see Example 1) and the cells were injectedintraventricularly to homozygous shiverer mice. One day old shi/shi pupswere anesthetized by brief hypothermia and received an injection of 10⁵cells in 2 μl, over a period of 3 minutes, into the brain thirdventricule, using a Hamilton 10 μl syringe. The pups were then warmedand returned to their mothers. Four weeks following transplantation, theanimals were given a pentobarbital anesthesia and killed by aorticperfusion with 4% PFA in 0.1 M phosphate buffer, pH 7.4. The brains werecryoprotected in phosphate-buffered 30% sucrose overnight. Serial 20μm-thick coronal cryostat sections were prepared from each brain andwere mounted on superfrost plus slides (Erie Scientific, Portsmouth,N.H.). Sections were thereafter immunostained with antibodies to themyelin protein MBP (red), human nuclear antigen (green) and stained withdapi (blue) to visualize nuclei, following the same procedures asdescribed in Example 2.

Ex vivo transplantation—Ex vivo transplantation of the human ES-NSc onshiverer brain slices maintained as organotypic cultures, was effectedaccording to the method described for murine ES-NSc (Zhang et al.,2006).

Electron microscopy—Brain slices were fixed in 2.5% glutaraldehyde and4% PFA in phosphate buffer (PB) 0.4 M, pH 7.4 for 12 hr, thencryoprotected in phosphate-buffered 30% sucrose overnight, and 20μm-thick coronal sections were prepared from various regions. Thesections were washed in 0.1 M Cacodylate buffer pH 7.4 and postfixedwith 1%. osmium tetroxide in the same buffer for 1-2 hours at RT. Afteren bloc staining with 2% uranylacetate in water for 1 hour at RT theslices were dehydrated in graded ethanol solutions and embedded ingraded Epon 812, as previously described (Shinder and Devor 1994).Ultrathin sections (70-90 nm thickness) were prepared withUltramicrotome Leica UCT, analyzed in a Philips Tecnai 12 TransmissionElectron Microscope at 120 kV and digitized with Megaview III CCD camerausing AnalySIS software.

Results

The effect of noggin on HuES-NSc was examined in brain sections ofShiverer mice transplanted with HuES-NSc. Results show that cellstreated with noggin (FIGS. 4 b-d) exhibit more MBP stained fibers thancells transplanted alone (FIG. 4 a). Quantitative analysis indicatesthat there is a significant 3.5-4 fold increase in the number of MBPfibers when noggin is added (FIG. 4 e). The fate of the injected humancells is illustrated FIGS. 4 c and f, which show MBP and nuclear dapistain of the same brain area. The area includes part of the thirdventricule (v) and MBP can be seen accumulating along the ventriculewalls (arrow) and more importantly in the brain parenchyma (arrowhead)where fibers of various lengths are observed over a large area (FIG. 4c). FIG. 4 g shows the same field stained for Human Nuclear Antigen(HNA, green) confirming the human origin of the myelinating cells. FIG.4 d shows an adjacent field (denoted by star in FIG. 4 f). These datademonstrate that the transplanted human cells produce myelin fibers overextended areas of the brain in the recipient animal.

Ex vivo transplantation of human ES-NSc—The myelinating capacity of thehuman ES-NSc was also demonstrated by ex-vivo transplantation onshiverer brain slices maintained as organotypic cultures. This methodallows measuring the myelinating capacity of the implanted cells morerapidly than in vivo injections. Two weeks after implantation ofHuES-NSc in the hippocampal region of a shiverer mouse brain slice,numerous MBP stained fibers were seen in an extended area of theentorhinal cortex (FIG. 5 a; FIG. 5 b shows the brain tissue nuclei withdapi-stain). At higher magnification, FIG. 5 c shows formation of densearrays of MBP-stained fibers, some of extended length (as indicated byarrows). Some of the fibers were a few hundred microns long (arrows),and dense arrays of similarly oriented and rather thick fibers werefound (FIG. 5 c). Few individual MBP⁺ cells could be seen. Electronmicroscopy confirmed that compacted myelin sheaths, with major denselines, were formed in the brain tissue transplanted with thenoggin-treated huES-NS cells (FIG. 5 e,f) contrasting with thedysmyelinated appearance of the shi/shi brain (FIG. 5 d). Such figuresof myelin were not seen with huES-NS cells untreated by noggin (whichwere like in FIG. 5 d). The noggin treatment markedly enhanced theoverall myelinating capacity, as quantitated by measuring the extent ofMBP staining and the mean length of MBP⁺ fibers formed (FIG. 5 g).

Taken together, these results establish that the procedure described inthe present invention yields human OP cells that can be expanded beforeengraftment and have the capacity to migrate and regenerate myelinfibers in brain of a dysmyelinated animal. The addition of noggin duringthe expansion stimulates the function of these OP cells to remyelinate.

Example 5 Derivation of Neurons from Human ES Cells

Materials and Experimental Procedures

Neuron generating culture—To attempt producing neurons from the human EScells, a neuron-generating culturing procedure was devised, based on theculturing procedure described in Example 1, wherein step M (adherence tomatrigel-coated plates) was omitted. Step R (treatment with retinoicacid) was done as in Example 1 but at the end of step S (neuroglialsphere ripening) the cells (dissociated by collagenase) were directlyplated on poly-ornithine and fibronectin (PO-FN)-coated tissue cultureplates (as described in Example 3. step D1) and cultured in ITTSSPP/B27with EGF and bFGF (10 ng/ml each), alternating with EGF alone (20ng/ml), for 10 days. Thereafter, the cells were passaged (1:2) by HBSSwhile scraped with a rubber policeman and the passage 2 cells werefurther cultured in the same conditions for an additional 6 days,afterwhich the medium was changed to N2/B27 medium and the same growthfactor regimen continued. For passage 3, the cells were dissociated withtrypsin, plated 2 days with ITTSPP/B27, EGF 20 ng/ml, and then mediumchanged again to N2/B27, with EGF plus bFGF (10 ng/ml each) for 4 days.For passage 4, the cells were dissociated by trypsin, replated in N2/B27with EGF plus bFGF for 2 days and then growth factors were removed, butlaminin, vitamin C and noggin (50 ng/ml) were added (as in step F3 ofExample 3). After 14 days the cells were fixed and stained. In anadditional culturing procedure, 100 ng/ml Sonic Hedgehog (Shh; R&DSystems, Minneapolis, Minn.) was added in step R.

Results

As indicated in Example 2, the huES-derived neurospheres (NS) culturedfor 4 days on the adherent matrigel substrate (step M, as described inExample 1) formed a network of neurons, but these tended to disappearafter prolonged culture on matrigel. To attempt producing neurons fromthe human ES cells, a neuron-generating culturing procedure was devised.The cultures contained neurons with long, connecting, processes(tubulin-βIII stain, FIG. 6 a), indicating that these conditions areappropriate to develop neurons from the neurospheres. GFAP stainindicated that the cultures also contained astrocytes, but nooligodendrocytes developed from the O4 stained cells, which were roundand appeared degenerating (not shown).

Sonic Hedgehog (Shh) is a factor made in the ventral spinal cord andplays an important role in inducing defined types of neurons as well asoligodendrocyte precursors (Marti and Bovolenta, 2002). When Shh wasadded in step R and the neuron-generating procedure described here wasapplied, there was an increase in the density of neuron network obtained(FIG. 6 b). However, no oligodendrocyte developed even with Shh (notshown). Therefore, the described procedure can be useful to obtainpopulations of human neurons from huES cells, without the presence ofoligodendrocytes.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications and GenBank Accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or GenBank Accession numberwas specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES (Other References are Cited Throughout the Document)

-   Amit, M. and Itskovitz-Eldor, J., 2002. “Derivation and spontaneous    differentiation of human embryonic stem cells.” J. Anat. 200,    225-232.-   Amit, M., Shariki, C., Margulets, V. and Itskovitz-Eldor, J., 2004.    “Feeder layer- and serum-free culture of human embryonic stem    cells.” Biol Reprod 70, 837-45.-   Bain, G., Kitchens, D., Yao, M., Huettner, J. E. and Gottlieb, D.    I., 1995. “Embryonic stem cells express neuronal properties in    vitro.” Dev Biol 168, 342-57.-   Brustle, O., Jones, K. N., Learish, R. D., Karram, K., Choudhary,    K., Wiestler, O. D., Duncan, I. D. and McKay, R. D., 1999.    “Embryonic stem cell-derived glial precursors: a source of    myelinating transplants.” Science 285, 754-6.-   Cao, Q., Benton, R. L. and Whittemore, S. R., 2002. “Stem cell    repair of central nervous system injury.” J Neurosci Res 68, 501-10.-   Chandran, S. and Compston, A., 2005. “Neural stem cells as a    potential source of oligodendrocytes for myelin repair.” J Neurol    Sci 233, 179-81.-   Foster, L. M., Landry, C., Phan, T. and Campagnoni, A. T., 1995.    “Conditionally immortalized oligodendrocyte cell lines migrate to    different brain regions and elaborate ‘myelin-like’ membranes after    transplantation into neonatal shiverer mouse brains.” Dev Neurosci    17, 160-70.-   Glaser, T., Perez-Bouza, A., Klein, K. and Brustle, O., 2005.    “Generation of purified oligodendrocyte progenitors from embryonic    stem cells.” Faseb J 19, 112-4.-   Gokhan, S., Marin-Husstege, M., Yung, S. Y., Fontanez, D.,    Casaccia-Bonnefil, P. and Mehler, M. F., 2005. “Combinatorial    profiles of oligodendrocyte-selective classes of transcriptional    regulators differentially modulate myelin basic protein gene    expression.” J Neurosci 25, 8311-21.-   Goldman, S., 2005. “Stem and progenitor cell-based therapy of the    human central nervous system.” Nat Biotechnol 23, 862-71.-   Grinspan, J., 2002. “Cells and signaling in oligodendrocyte    development.” J Neuropathol Exp Neurol 61, 297-306.-   Gumpel, M., Baumann, N., Raoul, M. and Jacque, C., 1983. “Survival    and differentiation of oligodendrocytes from neural tissue    transplanted into new-born mouse brain.” Neurosci Lett 37, 307-11.-   Itsykson, P., Ilouz, N., Turetsky, T., Goldstein, R. S., Pera, M.    F., Fishbein, I., Segal, M. and Reubinoff, B. E., 2005. “Derivation    of neural precursors from human embryonic stem cells in the presence    of noggin.” Mol Cell Neurosci 30, 24-36.-   Lachapelle, F., Gumpel, M., Baulac, M., Jacque, C., Duc, P. and    Baumann, N., 1983. “Transplantation of CNS fragments into the brain    of shiverer mutant mice: extensive myelination by implanted    oligodendrocytes. I. Immunohistochemical studies.” Dev Neurosci 6,    325-34.-   Liu, S., Qu, Y., Stewart, T. J., Howard, M. J., Chakrabortty, S.,    Holekamp, T. F. and McDonald, J. W., 2000. “Embryonic stem cells    differentiate into oligodendrocytes and myelinate in culture and    after spinal cord transplantation.” Proc Natl Acad Sci USA    97,6126-31.-   Marti, E. and Bovolenta, P., 2002. “Sonic hedgehog in CNS    development: one signal, multiple outputs.” Trends Neurosci 25,    89-96.-   McDonald, J. W., Liu, X. Z., Qu, Y., Liu, S., Mickey, S. K.,    Turetsky, D., Gottlieb, D. I. and Choi, D. W., 1999. “Transplanted    embryonic stem cells survive, differentiate and promote recovery in    injured rat spinal cord.” Nat Med 5, 1410-2.-   McKay, R., 1997. “Stem cells in the central nervous system.” Science    276, 66-71.-   Mehler, M. F., Mabie, P. C., Zhang, D. and Kessler, J. A., 1997.    “Bone morphogenetic proteins in the nervous system.” Trends Neurosci    20, 309-17.-   Mehler, M. F., Mabie, P. C., Zhu, G., Gokhan, S. and Kessler, J.    A., 2000. “Developmental changes in progenitor cell responsiveness    to bone morphogenetic proteins differentially modulate progressive    CNS lineage fate.” Dev Neurosci 22, 74-85.-   Nistor, G. I., Totoiu, M. O., Haque, N., Carpenter, M. K. and    Keirstead, H. S., 2005. “Human embryonic stem cells differentiate    into oligodendrocytes in high purity and myelinate after spinal cord    transplantation.” Glia 49, 385-96.-   Pfeiffer, S. E., Warrington, A. E. and Bansal, R., 1993. “The    oligodendrocyte and its many cellular processes.” Trends Cell Biol    3, 191-7.-   Powers, J. M. and Rubio, A., 1995. “Selected leukodystrophies.”    Semin Pediatr Neurol 2, 200-10.-   Reubinoff, B. E., Itsykson, P., Turetsky, T., Pera, M. F.,    Reinhartz, E., Itzik, A. and Ben-Hur, T., 2001. “Neural progenitors    from human embryonic stem cells.” Nat Biotechnol 19, 1134-40.-   Roach, A., Takahashi, N., Pravtcheva, D., Ruddle, F. and Hood,    L., 1985. “Chromosomal mapping of mouse myelin basic protein gene    and structure and transcription of the partially deleted gene in    shiverer mutant mice.” Cell 42, 149-55.-   Rogister, B., Ben-Hur, T. and Dubois-Dalcq, M., 1999. “From neural    stem cells to myelinating oligodendrocytes.” Mol Cell Neurosci 14,    287-300.-   Schachner, M., Kim, S. K. and Zehnle, R., 1981. “Developmental    expression in central and peripheral nervous system of    oligodendrocyte cell surface antigens (O antigens) recognized by    monoclonal antibodies.” Dev Biol 83, 328-38.-   Slutsky, S. G., Kamaraju, A. K., Levy, A. M., Chebath, J. and Revel,    M., 2003. “Activation of myelin genes during transdifferentiation    from melanoma to glial cell phenotype.” J Biol Chem 278, 8960-8.-   Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A.,    Swiergiel, J. J., Marshall, V. S. and Jones, J. M., 1998. “Embryonic    stem cell lines derived from human blastocysts.” Science 282,    1145-7.-   Warrington, A. E., Barbarese, E. and Pfeiffer, S. E., 1993.    “Differential myelinogenic capacity of specific developmental stages    of the oligodendrocyte lineage upon transplantation into    hypomyelinating hosts.” J Neurosci Res 34, 1-13.-   Yandava, B. D., Billinghurst, L. L. and Snyder, E. Y., 1999.    ““Global” cell replacement is feasible via neural stem cell    transplantation: evidence from the dysmyelinated shiverer mouse    brain.” Proc Natl Acad Sci USA 96, 7029-34.-   Zhang, P., Chebath, J., Lonai, P. and Revel, M., 2004. “Enhancement    of oligodendrocyte differentiation from murine embryonic stem cells    by an activator of gp 130 signaling.” Stem Cells 22, 344-54.-   Zhang, P. L., Izrael, M., Ainbinder, E., Ben-Simchon, L.,    Chebath, J. and Revel, M., 2006. “Increased myelinating capacity of    embryonic stem cell derived oligodendrocyte precursors after    treatment by interleukin-6/soluble interleukin-6 receptor fusion    protein.” Mol Cell Neurosci 31, 387-98.-   Zhang, S. C., Ge, B. and Duncan, I. D., 2000. “Tracing human    oligodendroglial development in vitro.” J Neurosci Res 59, 421-9.-   Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O. and Thomson, J.    A., 2001. “In vitro differentiation of transplantable neural    precursors from human embryonic stem cells.” Nat Biotechnol 19,    1129-33.

1. A method of generating neural and glial cells, the method comprisinggrowing human embryonic stem cells under conditions which inducedifferentiation of said human embryonic stem cells into the neural andglial cells, said conditions comprising the presence of retinoic acidand an agent capable of down-regulating Bone Morphogenic Proteinactivity.
 2. The method of claim 1, wherein when said cells are glialcells, conditions comprise: (a) growing said human embryonic stem cellsin the presence of retinoic acid, under conditions which allow formationof neurospheres; and (b) contacting said neurospheres with an agentcapable of down-regulating Bone Morphogenic Protein activity, therebygenerating said glial cells.
 3. (canceled)
 4. The method of claim 1,wherein when said cells are neurons, said conditions comprise: (a)growing said human embryonic stem cells in the presence of retinoic acidunder conditions which allow the formation of neurospheres; and (b)contacting said neurospheres with an agent capable of down-regulatingBone Morphogenic Protein activity under conditions which allow neuronalcells generation, said conditions comprising Sonic Hedgehog (Shh),thereby generating said neurons. 5-7. (canceled)
 8. An isolatedpopulation of cells comprising human cells wherein, (i) at least N % ofsaid human cells comprise at least one mature oligodendrocyte phenotype;and (ii) at least M % of said human cells comprise at least one stemcell phenotype. 9-12. (canceled)
 13. The cells of claim 8, wherein saidoligodendrocyte comprises a precursor oligodendrocyte phenotype. 14-17.(canceled)
 18. The cells of claim 13, wherein said precursoroligodendrocyte phenotype comprises a marker expression selected fromthe group consisting of PDGF-receptor, O4 sulfatide marker,galactocerebrosides (O1, GalC), Nkx2.2, Sox10, oligodendrocyte specificprotein (OSP), myelin-associated glycoprotein (MAG), 2′,3′-cyclicnucleotide-3′-phosphodiesterase (CNP), glutathione-S-transferase (GST),adenomatous polyposis coli (APC); myelin oligodendrocyte glycoprotein(MOG), CNPase, MOSP and Oligodendrocyte NS-1. 19-22. (canceled)
 23. Themethod of claim 1 wherein the glial cells comprise matureoligodendrocytes which comprise a mature oligodendrocyte phenotype. 24.The method of claim 23, wherein said mature oligodendrocyte phenotypecomprises a mature oligodendrocyte marker expression.
 25. The method ofclaim 24, wherein said mature oligodendrocyte marker is selected from agroup consisting of PLP, MBP, MAG and MOG. 26-27. (canceled)
 28. Themethod of claim 2, further comprising forming stem cell aggregates priorto step (a).
 29. The method of claim 1, wherein said conditions furthercomprise culturing said stem cells in the presence of a growth factor.30. The method of claim 29, wherein said growth factor is selected froma group consisting of bFGF and EGF.
 31. The method of claim 30, whereina concentration of said EGF comprises a range of 10-40 ng/ml.
 32. Themethod of claim 2, wherein said conditions further comprise culturingsaid neurospheres on an adherent substrate following step (a).
 33. Themethod of claim 4, wherein said conditions further comprise culturingsaid neurospheres on a cationic substrate following step (a). 34-36.(canceled)
 37. The method of claim 1, wherein a concentration of saidretinoic acid comprises a range of 1-50 μM.
 38. (canceled)
 39. Themethod of claim 2, wherein said step (a) is effected for 20-30 days. 40.The method of claim 2, wherein said step (b) is effected for 6-10 days.41. (canceled)
 42. The method of claim 1, wherein said agent is noggin.43. The method of claim 42, wherein a concentration of said noggincomprises a range of 10-100 ng/ml.
 44. The method of claim 2, whereinsaid step (b) is effected at least in part in the absence of growthfactors.
 45. The method of claim 44, wherein said step (b) is effectedin the presence of laminin and vitamin C.
 46. The method of claim 2,further comprising: dissociating cells of said neurospheres; andpassaging said dissociated cells; following step (a) and/orconcomitantly with step (b).
 47. The method of claim 46, wherein saidpassaging is effected every 8-10 days.
 48. The method of claim 2,further comprising isolating a glial cell subpopulation of interestfollowing step (b).
 49. (canceled)
 50. The method of claim 4, furthercomprising isolating said neurons following step (b). 51-58. (canceled)